J Mol Evol (1992) 35:156-180

Journal of Molecular Evolution @ Springer-Verlag New York Inc. 1992

Synthesis Evolutionary Consequences of Nonrandom Damage and Repair of Chromatin Domains* Teni Boulikas Linus Pauling Institute of Science and Medicine and Institute of Molecular Medical Sciences, 440 Page Mill Road, Palo Alto, CA 94306, USA

Summary.

Someevolutionaryconsequencesofdifferent rates and trends in D N A damage and repair are explained. Different types o f D N A damaging agents cause n o n r a n d o m lesions along the DNA. T h e type o f D N A sequence motifs to be preferentially attacked depends upon the chemical or physical nature o f the assaulting agent and the D N A base composition. Higher-order c h r o m a t i n structure, the n o n r a n d o m nucleosome positioning along the DNA, the absence o f nucleosomes from the p r o m o t e r regions o f active genes, curved DNA, the presence o f sequence-specific binding proteins, and the torsional strain on the D N A induced by an increased transcriptional activity all are expected to affect rates o f damage o f individual genes. Furthermore, potential Z - D N A , H - D N A , slippage, and cruciform structures in the regulatory region o f some genes or in other genomic loci induced by torsional strain on the D N A are m o r e prone to modification by genotoxic agents. A specific actively transcribed gene may be preferentially damaged over nontranscribed genes only in specific cell types that maintain this gene in active c h r o m a t i n fractions because o f (1) its decondensed c h r o m a t i n structure, (2) torsional strain in its D N A , (3) absence o f nucleosomes from its regulatory region, and (4) altered nucleosome structure

* Dedicated to Emile Zuckerkandl and Costas Kastritsis

Abbreviations: MNNG, N-methyl-N'.nitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; ENU, N-ethyl-N-nitrosourea; HO-AAF, N-hydroxy-2-acetylaminofluorene; DMBA, 7,12-dimethylbenz[a]anthracene; cis-DDP, cis-diaminedichloroplatinum (II); hsp, heat shock protein; MHC, major histocompatibility complex; CHO, Chinese hamster ovary; ADA, adenosine deaminase; DHFR, dihydrofolate reductase; HPRT, hypoxanthine (guanine) phosphoribosyltransferase; GPDH, glyceraldehyde 3' phosphate dehydrogenase; HMG, high mobility group

in its coding sequence due to the presence o f modified histones and H M G proteins. The situation in this regard o f germ cell lineages is, o f course, the only one to intervene in evolution. Most lesions in D N A such as those caused by U V or D N A alkylating agents tend to diminish the G C content ofgenomes. Thus, D N A sequences not b o u n d by selective constraints, such as pseudogenes, will show an increase in their A T content during evolution as evidenced by experimental observations. On the other hand, transcriptionally active parts m a y be repaired at rates higher than inactive parts o f the genome, and proliferating cells m a y display higher repair activities than quiescent cells. This might arise from a tight coupling o f the repair process with both transcription and replication, all these processes taking place on the nuclear matrix. Repair activities differ greatly a m o n g species, and there is a good correlation between life span and repair a m o n g mammals. It is predicted that genes that are transcriptionally active in germ-cell lineages have a lower m u t a t i o n rate than bulk DNA, a circumstance that is expected to be reflected in evolution. Exception to this rule might be genes containing potential Z - D N A , H - D N A , or cruciform structures in their coding or regulatory regions that appear to be refractory to repair. This study supports the molecular clock hypothesis when applied to one gene within a group o f related species and contends that evolutionary rates might vary between genes and gene segments not only as a result o f differences in selective constraints but also as a result o f differences in the rate o f damage minus rate o f repair a m o n g different segments o f c h r o m a t i n DNA.

Key words:

D N A damage -- D N A repair -- Chromatin -- Evolution -- Nucleosomes -- Nuclear ma-

157 trix -- Active genes -- Z-DNA -- Sperm -- Mutation -- Molecular clock

Introduction Nucleic acids are reactive molecules and therefore undergo structural changes under the influence of chemical attack. The rate of mutations and hence, according to the neutral theory, the rate of fixation of base substitutions during evolution depends on the rate of introduction of lesions into DNA and their rate of repair. Repair of damaged DNA is an essential molecular process fundamental to the maintenance of life, evolved at an early time for the restoration of the original status of the genome. Repair mechanisms, operating in all organisms from bacteria to humans, contribute to the genetic stability of organisms. Nevertheless, most DNA repair processes are error prone. The lack of 100% efficiency in repair mechanisms has contributed to the genetic diversity on earth and in the generation of variants in populations within species (Wintersberger 1991). Natural populations are able to survive unexpected catastrophes only because they allow variability among individuals, some of which have the fortuitous ability to cope with unexpected environmental stress (Luria and Delbriick 1943). Damage to DNA, thus, induces evolution (Echols 1981; Wintersberger 1984; Cairns et al. 1988; Klein et al. 1989, 1990). Several recent studies have established that damage on intact cells (or isolated sequences) is not randomly distributed over segments ofchromatin DNA or even between the transcribed and nontranscribed strands of the same gene (e.g., Vrieling et al. 1989, 1991; McGregor et al. 1991). Data showing preferential repair of active compared with inactive genes (see section II) would result in a nonuniform distribution of mutation rates among various segments of DNA. Molecular evolution intervenes and can change the trend of the incurred nucleotide change by random drift or natural selection. The final trend of damage, repair, and molecular evolution will still result in a nonrandom distribution of mutation rates along the chromatin fiber as noted by Wolfe et al. (1989). Rates ofnonsynonymous substitution among genes may vary by as much as 1000-fold as, for example, between the highly conserved histone H4, H3, and ubiquitin genes on one hand and the apolipoprotein, immunoglobulin, and interferon genes on the other hand. However, rates of synonymous substitution may range only about fivefold, i.e., from about 2 to about 9 substitutions per nucleotide per 109 years within coding regions of genes (see Li and Graur 1991). Thus, rates of nonsynonymous substitution seem to be determined by the neutral drift

or natural selection, which in turn depend upon the functional constraints imposed by the degree of freedom for altering the amino acid composition of a protein without impairing its function. Here, we shall examine the factors that affect rates of damage, rates of repair, and speculate on their evolutionary implications.

I. Damage

1. Preferentially Damaged Bases The distinct preference of most chemicals for attacking and modifying one base of DNA over the other three has been well established. The most frequently attacked of all four bases seems to be guanine. Indeed, the 06 position in guanines is preferentially alkylated by MNNG, MNU, ENU (Singer 1976), and alkylnitrosamines (Pegg et al. 1984). Guanine is also preferentially methylated at its N 7 position by dimethylsulfate (Reiner and Zamenhof 1957; Mirzabekov et al. 1977) and methylmethane sulfonate (Loveless 1969). 7-Methylguanine can correctly pair with cytosine during DNA replication and is not mutagenic (Ludlum 1970). However, N 7methylation at a single guanine within DNA sequences that specifically interact with sequence-specific proteins might strongly inhibit protein binding to its cognate DNA sequence and thus change the epigenetic information; for example, a rupture in protein-DNA interaction has been demonstrated to take place at the octamer motif within the immunoglobulin enhancer region following methylation (Sen and Baltimore 1986). Antitumor chloroethylating agents (Hartley et al. 1986) and nitrogen mustards (Mattes et al. 1986) that alkylate the N v position of guanine display a distinct DNA base composition selectivity. Adenine is attacked to a lesser extent by these DNA alkylating agents, whereas cytosines and thymidines are almost unreactive. Contiguous stretches of two or more Gs or As are preferentially attacked over a single G or A by alkylating agents; this might explain the preferential attack of both AT- and GC-rich satellites by ENU and M N N G observed by Durante et al. (1989). Aflatoxin displays a distinct base preference (Foster et al. 1983; Muench et al. 1983). HO-AAF preferentially forms adducts with guanines (Beranek et al. 1982). DMBA preferentially binds to adenines and guanines (Dipple et al. 1983). Similarly, benzpyrene-7,8-diol-9,10-epoxide binds to individual G residues with preference for long poly(dG) stretches (Boles and Hogan 1986). The middle Gs in G runs seem to be preferentially alkylated (Mattes et al. 1986). Poly(dG) stretches are present in the promoter region of several genes such as the chicken ¢/-globin (Clark et al. 1990) and the gastrin genes

158 (Merchant et al. 1991). Cis-DDP, an antitumor agent of major clinical importance, forms interstrand crosslinks between the N 7 atoms of two alternating G residues, preferentially in GC dinucleotides (Lemaire et al. 1991). The dinucleotide CpG is the most frequent "hot spot" for spontaneous germ-line mutations in the human genome (Cooper and Krawczak 1989). In part, this might result from the inability of potential Z-DNA sequences to be repaired (Boiteux et al. 1985). The preferential modification of one base by specific chemicals, some of which are of special interest due to their potential as naturally occurring mutagens, has been exploited to develop the chemical method of DNA sequencing (Maxam and Gilbert 1977). Furthermore, several reagents were found with a distinct preference for a specific base in methods aimed at detecting Z-DNA. For example, hydroxylamine reacts very strongly with cytosines at junctions between B- and Z-DNA (Johnston and Rich 1985) and in Z-Z junctions (Johnston et al. 1991). Similarly, chloracetaldehyde is hyperreactive to B-Z junctions (Kohwi 1989). Diethylpyrocarbonate is hyperreactive with all purines in alternating purine-pyrimidine sequences (Johnston and Rich 1985). Osmium tetroxide displays a preference for exposed thymines in B-Z junctions (Nejedly et al. 1985).

2. The Effect of Base Composition on Rates of Damage Neighboring bases seem to affect rates of damage. For example, changing a single DNA base pair 12 bp away from a mutagenesis site in bacteriophage T4 lowers the probability of conversion of this site to 2-aminopurine eightfold (Sugino and Drake 1984). Based on a systematic study of UV-induced lesions within a specific gene inserted into a shuttle vector plasmid and transfected into xeroderma pigmentosum group A and normal human fibroblasts, Brash and collaborators (1987) have shown that such lesions varied by as much as 80-fold at different dipyrimidine sites within the gene. Similar conclusions were reached by other laboratories (ProticSabljic et al. 1986; Drobetsky et al. 1987; Armstrong and Kunz 1990; Smerdon and Thoma 1990; Pfeifer et al. 1991). In addition to damage directly at the site of mutation, the potential for nearby oppositestrand damage may be important for determining the mutability of a site (Schaaper et al. 1987). Thus, the mutation frequency in particular genes seems to be determined primarily by some structural features of their DNA; even oncogene activation might take place because of some DNA base composition peculiarities present within them (Brash et al. 1987). It remains to be shown whether preferred sites of

DNA damage in some coding regions of genes coincide with the less conserved regions in some proteins that are under a low rate of functional constraints and evolve more rapidly than average genome sites (Kimura and Ohta 1973; Kimura 1989; Li and Graur 1991; Zuckerkandl 1992). Irradiationof cells with UV induces cyclobutane pyrimidine dimers by formation of two covalent bonds between adjacent TT, CT, TC, and CC residues on the same strand of DNA; at a lower frequency, induction of pyrimidine (6-4) pyrimidone photoproducts takes place by formation of a covalent bond between the carbon 6 and carbon 4 of adjacent pyrimidines (Setlow and Setlow 1963; Brash and Haseltine 1982; Brash 1988). The (6-4) photoproducts, although produced 10 times less frequently than pyrimidine dimers, seem to be mutagenic (Glickman et al. 1986; Mitchell and Nairn 1989). Cyclobutane pyrimidine dimers are the major form of premutational DNA damage caused by UV (Armstrong and Kunz 1990) and are mutagenic in mammalian cells (Protic-Sabljic et al. 1986). We do not know whether h o m o p y r i m i d i n e stretches will be mutated more heavily by UV than dipyrimidines. Whenever poly(dT) tracts are present, thymidine dimers are more readily formed at the 3' terminal TT site of the poly(dT) tract following irradiation of DNA in vitro (Lyamichev 1991). However, homopyrimidine tracts have the potential of forming triple helices (e.g., Johnston 1988) and to be attacked at higher rates by some chemicals compared to typical B-DNA. This is due to the presence of a single-stranded loop structure (Kohwi 1989). Homopyrimidine repeats are characteristic elements of several genes such as of the human ETS-2 oncogene, a human U1 gene, the murine immunoglobin Ca switch region, Drosophila heat shock genes, human U2 genes, the chicken/3A globin gene, and many other genes (reviewed by Boulikas 1991). According to Birnboim (1978), 0.5% of the mouse genome consists of homopyrimidine tracts over 20 bases in length. The notion that base composition affects rates of DNA damage can be extended to individual genes. It may be possible to find differences in rates of damage between genes, between two segments of the same gene, as well as between the two strands of a specific gene segment. For example, benzopyrene seems to cause lesions preferentially in the nontranscribed over the transcribed strand of the HPRT gene (Chen et al. 1990). UV-induced mutations in yeast seem to arise preferentially at dipyrimidine sequences in the transcribed strand of the SUP4-o gene (Armstrong and Kunz 1990). The frequency of UV-induced lesions in the nontranscribed strand in a 180-nucleotide region (NC + region) encoding a highly conserved protein domain in the lac I gene

159 in Escherichia coli was higher than in the transcribed strand (Koehler et al. 1991). HO-AAF activated H-ras by a specific C -~ A mutation in codon 61 in seven out of seven mouse liver tumors studied (Wiseman et al. 1986). MNU activation of the H-ras gene in rat mammary tumors shows complete bias for causing G -~ A base changes of codon 12 in 61 out of 61 tumors examined (Barbacid 1987).

preferably in linker regions (e.g., Seidman et al. 1983; Sogo et al. 1986) and induce sharp bends or kinks in the DNA helix. The bulky adduct guanine-8-aminofluorene causes DNA deletions (Mitchell and St6hrer 1986). Ionizing radiation produces apurinic/apyrimidinic sites as well as strand breaks (Giloni et al. 1981) and causes base substitutions, frameshifts, and small deletions in mammalian cells (Grosovsky et al. 1988).

3. Damaging Agents and Type of Mutations Different types of mutagens induce different types of mutations in the DNA. Both pyrimidine dimers and (6-4) photoproducts caused by UV are responsible for C ~ T transitions during replication of DNA (Vrieling et al. 1989). Human cells repair virtually all (6-4) photoproducts within about 6 h after damage, whereas cyclobutane pyrimidine dimers are removed at slower rates (Mitchell et al. 1985). M N N G and MNU preferentially methylate the 06 position of guanines; this does not allow formation of the proper GC pair during replication, and thus both M N N G and MNU are potent mutagens (Singer 1976). O6-methylated guanine pairs mainly with T (Snow et al. 1984) to give G . C -~ A . T mutations equivalent to G -~ A transitions (Loechler et al. 1984; Richardson et al. 1987). The consequence of a single G -~ A transition occurring at a specific site along the DNA can have important consequences. For example, it may result in a higher level of expression of a gene that may lead to the induction of papillomas in mouse skin (Brown et al. 1990). Similarly, bromodeoxyuridine (BrdU) may induce G A transitions; this is caused by misincorporation of BrdU opposite guanine occurring almost exclusively in sequences with two adjacent guanine residues and involving the one in the 3' side (Davidson et al. 1988). 3-Methylcholanthrene may induce both G T and A ~ T transversions (Brown et al. 1990). HOAAF preferentially forms adducts with guanines and induces G. C -, T. A mutations (Beranek et al. 1982). Aflatoxin B1 may cause G ~ T transversions that again, when occurring at a specific position within a tumor suppressor gene, may lead to hepatocellular carcinomas (Hsu et al. 1991). Reactive oxygen species produce strand breaks, base loss or damage, and fragmentation of the deoxyribose moiety (Hutchinson 1985; Cross et al. 1987). Ionizing radiation causes deletions of hundreds of base pairs or more, inversions and other DNA rearrangements, and a smaller number of point mutations (reviewed by Thacker 1985; Breimer 1988). In addition, ionizing radiation induces loss of DNA bases and strand breaks with 3' phosphate or 3' phosphoglycolate groups (Giloni et al. 1981; Hutchinson 1985). Acetylaminofluorene, psoralen, and benzpyrene form chemical adducts on DNA

4. Higher-Order Chromatin Structures Affect Rates of DNA Damage DNA in higher-order chromatin structures appears to be less accessible than DNA in decondensed chromatin to DNase I and micrococcal nuclease as well as to a variety of damaging agents (reviewed by Boulikas 1987a,b). Even mechanical breakage on DNA by sonication of nuclei renders DNA in higher-order chromatin structures intact under conditions where open chromatin sites coinciding with the DNase I hypersensitive sites are broken (Reneker and Brotherton 1991). An extreme example supporting the idea that higher-order chromatin structures are less accessible to damage arises from mature mammalian spermatocytes; due to a remarkable condensation of DNA brought about by the replacement of histones by protamines, sperm DNA is unreactive to benzpyrene (Balhorn et al. 1985) and inaccessible to intercalation by actinomycin D (Darzynkiewicz et al. 1969). The idea that higher-order chromatin structures might affect rates of damage has been previously discussed (Boulikas 1991, 1992).

5. The Nonrandom Nucleosome Positioning Along the DNA May Determine Rates of DNA Damage MAXIMA OF U V DAMAGE ALONG THE NUCLEOSOME D N A

As the DNA double helix wraps around the core histone octamer, histones interact electrostatically with phosphate groups in the major groove (Mirzabekov et aI. 1978). Because the core histone octamer interacts with the major groove of DNA at interrupted sites spaced about every 10.3 nucleotides, some mutagens may display a 10.3-nucleotide periodicity of damage along the nucleosome DNA. UV irradiation was shown to produce a 10.3-nucleotide pyrimidine dimer periodicity along nucleosomes; sites of high probability for pyrimidine dimer formation were at positions where the DNA strand is farthest from the histone surface (Jensen and Smerdon 1990). The 10.3-bp periodicity of UV damage will tend to give C -~ T substitutions and might provide the T-rich periodicity of curved DNA (Trifonov 1986;

160 Trifonov and Mengeritsky 1987). During evolution, UV irradiation may thus have caused a stabilization of the positions of nucleosomes along the DNA by creating periodic sites of preferential interaction with the nucleosome (Trifonov 1989). This periodicity therefore need not be explained by an effect of natural selection; rather, it is expected to be spontaneously formed once histone octamers are available in excess and saturate random DNA. Secondarily, a given interaction between core histone octamers and DNA may have become part of a functional macromoleeular interaction system and may then be subject to natural selection. It is of interest to realize that bases that occur with a periodicity of 10.4 may have been generated by external agents of "imprinting" whose action might be likened to the automatic and programmed etching of microchips. RATES OF DAMAGE OF LINKER VERSUS NUCLEOSOMAL D N A

DNA in nucleosomes seems to be more protected from damaging agents than linker or totally naked DNA. For example, psoralens display a distinct preference for intercalating into and cross-linking with the two strands in linker over nucleosomal DNA (Sogo et al. 1986). Similarly, benzpyrene 7,8diol-9,10-epoxide (Seidman et al. 1983) and anthracyclins, a family of antibiotics with antitumor activity (Cera et al. 1991), display a clear preference for binding to linker over nucleosomal DNA. DNA breaks caused by singlet oxygen occur preferentially at kinks located at 1.5 helical turns from the center of nucleosomal DNA (Hogan et al. 1987). Pyrimidine-pyrimidone (6-4) photoproducts induced by UV occur with a sixfold greater frequency in the linker than in core nucleosomal DNA (Mitchell et al. 1990). The nonrandom arrangements of core histone octamers along DNA strongly suggest that DNA sequences wrapped around core histone octamers might be attacked at lower rates than linker DNA by certain classes of carcinogens. The unique or preferential positions of nucleosomes along the two strands of DNA are primarily determined by the preference of core histone octamers to be centered around particular DNA sequences that bend easily (Thoma and Simpson 1985; Trifonov 1986; Bolshoy et al. 1991) and by specific DNA sequence landmarks such as potential Z-DNA or triple helical structures that exclude nucleosome f o r m a t i o n (Simpson and Kiinzler 1979; Kunkel and Martinson 1981; Prunell 1982). ABSENCE OF NUCLEOSOMES FROM REGULATORY REGIONS OF ACTIVE GENES AND RATES OF DAMAGE

DNA sequence-specific proteins including the trans-acting nuclear factors that bind to the en-

hancer and to the promoter region of genes as well as to the origins of replication seem to bend the DNA and to exclude nucleosomes from these regions. Well-established examples are the 400-bp nucleosome-free gap at the origin of replication of SV40 minichromosomes containing the binding site of T antigen (Varshavsky et al. 1979; Saragosti et al. 1980) and the removal of several nucleosomes by trans-acting protein factors from the promoter of a gene during its induction to transcription (A1mer et al. 1986; Richard-Foy and Hager 1987; Straka and Hrrz 1991). The protein-DNA cross-linking data of Nacheva and coworkers (1989) have confirmed the absence of histones from the promoter region of the transcriptionally active hsp70 gene in Drosophila. Furthermore, sequence-specific nonhistone proteins seem to serve as boundaries for nucleosome arrays (Simpson 1986; Travers 1987; Losa et al. 1990; Roth et al. 1990) and may thus determine, along with the DNA base composition (Trifonov and Mengeritsky 1987), which sequences will be wrapped around octamers and become more protected. Thus, promoter and enhancer regions as well as origins of replication should suffer damage at a higher rate than adjacent regions that bind nucleosomes. On the other hand, a significant proportion of nucleotides in these regions is expected to be conserved by natural selection because they need to bind specific proteins. Their preferential attachment to the nuclear matrix where repair takes place may ensure their preferential repair over the bulk genome (reviewed by Boulikas 1992). The base pair actually found at a given position will be an outcome of three processes: damage, repair, and natural selection. At a given nucleotide position, any one of the three may occur, not occur, or occur in combination. If natural selection does not occur, any substitution when unrepaired will spread in the population or be lost from the population by a random process (neutral drift). If natural selection does occur, unrepaired damage will be eliminated except for the small portion of advantageous mutations. If repair does not occur after damage, the new nucleotide may be eliminated or maintained either by natural selection or by random drift. The comparison between homologous (orthologous) sequences in different species does not permit one to judge of an average evolutionary rate of unrepaired damage because most of the substitutions attributable to unrepaired damage will be lost either by drift or by negative selection, whereas some (a much smaller proportion) will have been fixed by positive selection. Mutations within coding regions of genes have made a qualitatively large contribution to molecular evolution. Specific hot spots displaying a maximal

161 variation in nucleotide substitution have been found within genes. In several cases specific mutations that impart growth advantages like those specifically occurring at codon 12 of the H-ras gene appear to be responsible for the clonal expansion of the mutated cell, giving rise to cancer (Capon et al. 1983). This might be considered an analogue of the expansion of a subpopulation in a species carrying a mutation that offers an advantage in a changed environment (Dobzhansky 1970). Although this does not necessarily imply that mutations in the H-ras gene occur exclusively at codon 12 but rather that this particular mutation will cause an expansion in this cell population and will be selected and fixed in these cancer ceils, this particular cell expansion might be facilitated if this particular site were more prone to mutation than average genomic sites. Indeed, M N U is surprisingly biased in mutating this particular position of the H-ras oncogene: single doses of M N U caused a G -~ A base change in the H-ras gene in 61 of 61 tumors examined (reviewed by Barbacid 1987). The effect of the preferential location of such mutational hot spots in linker sequences rather than in nucleosomes or the binding of DNA sequencespecific proteins to such regions on molecular evolution has not been investigated.

6. Transcriptional Activity, Cell Type, and Cell Cycle Dependence of the Preferential Damage of Active Over Inactive Genes Unwinding of the right-handed DNA double helix during transcription generates positive torsional strain ahead and negative behind the RNA polymerases during their passage along the chromatin template (Giaever and Wang 1988; Tsao et al. 1989). Negative torsional strain on the DNA is known to induce unusual DNA structures such as Z-, lefthanded DNA (Johnston and Rich 1985), triple-helical, H-DNA (Kohwi 1989), and cruciform DNA structures (Panayotatos and Fontaine 1987; Frappier et al. 1989). An increase in Z-DNA in human HL-60 cells in culture determined with Z-DNA antibodies was found associated with an increase in the transcriptional activity and transcription-generated torsional strain (Wittig et al. 1991). Such unusual DNA structures, due to the singlestranded character at their boundaries with the typical B-DNA structure or at internal sites, are more easily attacked by chemicals (see section I, part 1). In addition, lesions in such unusual DNA structures might persist due to the inability of DNA repair enzymes to use them as substrates (Boiteux et al. 1985; Topal 1988). Potential Z-DNA, H-DNA, and cruciform structures are preferentially located within the promoter or other regulatory regions of several genes (reviewed by Boulikas 1991, 1992). Ex-

amples are the (TCCTC)28 motif present in the murine immunoglobin Ca switch region (Davie and Murphy 1990), the (CT)75 repeat in the promoter region of the human ets-2 oncogene (Mavrothalassitis et al. 1990), the GC-rich potential Z-DNA sequence in the 5' upstream regulatory region of the rat and human cartilage link protein gene (Rhodes et al. 1991), and the murine /3-major globin gene (Gilmour et al. 1984). It is reasonable to anticipate that specific types of DNA damaging agents might damage the unprotected nucleosome-free region of a gene promoter only in those cell types harboring this gene in a transcriptionally active chromatin structure for four reasons: because of (1) differences in chromatin condensation, (2) differences in the torsional strain in the DNA, (3) absence of nucleosomes from the regulatory region of active but not of inactive genes, and (4) altered structure of active compared with inactive nucleosomes, due to the presence of modified histones and HMGs (see below). The same gene might be less sensitive to the same type of agents in a different cell type not expressing this gene. In addition, the activity of DNA repair enzymes greatly depends upon the cell type (Planche-Martel et al. 1985; Belinsky et al. 1988). Proliferating cells such as cells in developing organs, embryos, or tumors have a much greater DNA repair capacity than terminally differentiated, nonproliferating cells (e.g., Liu et al. 1983). The preferential attack of the coding region of actively transcribed over transcriptionally inactive genes by mutagens has not been explored. Such differences might be found, because active genes, although containing nucleosomes in their coding regions (Einck and Bustin 1983; De Bernardin et al. 1986; Solomon et al. 1988), seem to harbor split nucleosomes (Lee and Garrard 1991), partially unfolded nucleosomes (reviewed by Csordas 1990), or constantly dissociating core histone octamers that reassociate with negatively supercoiled DNA immediately behind the transcription complex, as proposed by Clark and Felsenfeld (1991). In addition, nucleosomes in active chromatin seem to be enriched in acetylated, ADP-ribosylated, and ubiquitinated histones (reviewed by Boulikas 1992) as well as in H M G nonhistones (Dorbic and Wittig 1987). The protection of DNA by modified histones or HMGs in altered nucleosome structures toward its reaction with mutagens is not known. Mutations induced by UV in S-phase human cells were mainly located in the 3' end of the HPRT gene; however, UV-irradiation of G 1-phase cells showed an even distribution of the UV lesions along the HPRT gene (McGregor et al. 1991). Thus, gene segments might show a change in their vulnerability to mutagens during the cell cycle. Replicating DNA is preferentially damaged by

162 MNU in cells in culture (Cordeiro-Stone et al. 1982). This implies that genes in proliferating cells might be more prone to damage. However, the tight link between repair and replication (see below) and the higher repair capacities in proliferating versus quiescent cells, along with the proofreading mismatch repair ability of DNA polymerases might rapidly eliminate incurring lesions during replication.

II. Repair 1. Active Genes Are Repaired at Higher Rates than Inactive Genes

Numerous studies have demonstrated that active genes are repaired at higher rates than inactive genes. Higher-order chromatin structures impose a barrier to repair enzymes. Early data have demonstrated that DNA lesions in chromatin are not uniformly accessible to repair (Wilkins and Hart 1973; Bodell 1977; Cleaver 1977). The pioneering work of Hanawalt, Bohr, and coworkers (reviewed by Bohr et al. 1987; Hanawalt 1989; Boulikas 1991, 1992) has indeed shown that mammalian cells in culture are able to repair active genes at rates 2-100-fold higher than rates of repair in inactive genes. In most studies it was mainly the repair of UV-induced damage on the DNA, i.e., the removal of pyrimidine dimers and (6-4) photoproducts that was addressed. For example, UV damage in the actively transcribed and amplified D H F R gene was repaired faster than the bulk of the genome in CHO (Bohr et al. 1985; Ho et al. 1989) and in human cells (Mellon et al. 1986). These studies were extended in several other laboratories. A preferential repair ofpyrimidine dimers in the transcriptionally active c-myc locus over bulk DNA in B lymphoblasts was ascertained by Beecham et al. (1991). In Saccharomyces cerevisiae a 2.5-fold higher rate of repair of the active MA Ta locus as compared to the inactive H M L a locus was found by Terleth and collaborators (1989). Only 51% of the pyrimidine dimers were repaired in 24 h in the inactive 754 locus in confluent human fibroblasts (VH 16), whereas about 70% of the pyrimidine dimers were removed in 8 h from the active ADA gene (Venema et al. 1990b). Repair of pyrimidine dimers and (6-4) photoproducts in the very actively transcribed /3-actin and c-myc genes was about the same as repair rates in the moderately transcribed D H F R and GPDH genes; in turn, /3actin, c-myc, DHFR, and G P D H genes were more efficiently repaired than the nontranscribed c-mos and glutamic acid decarboxylase genes in mouse lymphoid cells in culture (Russev and Boulikas 1992). Thus, there seems to be a good correlation between transcriptional activity and repair efficiency for individual genes.

Active genes may differ from one another in their repair efficiencies. O6-methylguanine removal between the transcriptionally active c-neu and active c-myc oncogenes was found to differ substantially: whereas the c-neu gene was repaired in 24 h in rat lung epithelial cells in culture, no repair was detected in the c-myc gene (Chary et al. 1991). The above studies were extended by LeDoux and coworkers (1990) to the insulin gene in two rat insulinoma cell lines differing in the levels of insulin gene expression. Using methylnitrosourea rather than UV, a threefold higher efficiency in the repair of the insulin gene in the cell line that exhibited the higher levels of insulin gene expression was found. Using UV irradiation, Ramanathan and Smerdon (1989) found that repair rates were higher in a fraction of nucleosomes enriched in hyperacetylated histone H4. Presumably, this fraction represented transcriptionally active chromatin. Similarly, transcriptional activation of the metallothionein gene by heavy metals resulted in more efficient repair of UV-induced damage in CHO cells (Okumoto and Bohr 1987). Demethylation of DNA caused by treatment of cells in culture with 5-azacytidine assimilating the fully active demethylated early embryonic DNA state resulted in an increase in repair at the 3' end o f D H F R gene (Ho et al. 1989). These lines of evidence give additional support to the idea that active genes are repaired more efficiently than inactive genes or bulk DNA. A smaller number of studies has failed to show a difference in repair rates between active and inactive genes. For example, whereas UV damage was repaired faster in the D H F R gene compared to the overall genome in CHO cells (Bohr et al. 1985), repair of dimethylsulfate-induced damage on the DNA (mostly accounting for the repair of the N 7methylguanine) in the same cells occurred at comparable rates between the active D H F R gene and a nontranscribed downstream region (Scicchitano and Hanawalt 1989). Although the studies of Mellon et al. (1987), Smerdon and T h o m a (1990), and McGregor et al. (1991) have shown a higher repair rate of UV damage in the transcribed compared to the nontranscribed strand of DNA, the studies of Govan et al. (1990) suggested similar rates of repair between the two strands in the D H F R gene after UV damage using human B-lymphocytes. More data are required for drawing a conclusion on the preferential repair of the transcribing as compared to the nontranscribing strand. The studies of de Cock and collaborators (1991) have suggested that pyrimidine dimers from the actively transcribed Gart and Notch genes in Drosophila were removed at the same rate and to the same extent as pyrimidine dimers in the transcriptionally inactive white gene and in the genome over-

163 all. Evidently, all of the Drosophila genome is repaired efficiently (de Cock et al. 1991), whereas in mammalian cells a large part of the genome is repaired slowly (e.g., Bohr et al. 1985). The slowly repaired part of the mammalian genome might be associated with the late replication-associated facultative heterochromatin as opposed to the earlyreplicated euchromatin reflected in R- and G-banding patterns (Holmquist 1987, 1989). Such patterns have not been observed in Drosophila chromosomes (Steinemann 1981), and this was suggested by de Cock and coworkers (1991) to explain their finding that Drosophila repairs active and inactive genes at identical rates. However, Drosophila seems to repair overall DNA damage less efficiently than mammalian cells because DNA polymerase/3 involved in repair in mammals is not found in Drosophila (Filipski 1987). This finding cannot explain the lower mutation rate of Drosophila compared to mice (Russell 1956). In spite of some controversial findings, the bulk of the evidence shows convincingly that transcriptionally active genes in mammals, especially in rodents, are repaired faster than bulk DNA; in addition, the transcribed strand of an active gene appears to be repaired more efficiently than the nontranscribed strand.

2. Models Explaining Differences in Repair Rates among Genes Three models have been proposed to explain the preferential repair of active versus inactive genes. First, based on their studies on the repair of the lac operon locus in E. coli, Mellon and Hanawalt (1989) postulated that an R N A polymerase complex blocked at a lesion might be a recognition signal for repair. Second, the preferential repair of active over inactive genes may arise from their open, uncondensed chromatin structure thought to be more accessible to DNA repair enzymes (Hanawalt 1991; Russev and Boulikas 1992). Third, the preferential repair of active over inactive genes was proposed by Boulikas (1992) to arise from the need of damaged DNA to be attached to the nuclear matrix where the repair enzymes are preferentially located. This is schematically shown in Fig. 1. In such a case, actively transcribed genes are already attached to the nuclear matrix, and their repair is facilitated. Indeed, transcription, replication, and repair all seem to occur in association with the nuclear matrix (reviewed by Boulikas 1987a,b, 1991, 1992). Differences in repair efficiencies among organisms might arise from differences in the abundance and efficiency of the great number of DNA repair enzymes in their nuclei and from differences in the

proofreading ability of DNA polymerases in different organisms (see section II, part 4).

3. Differences in Repair Rates between the Transcribed and Nontranscribed Strands of the Same Gene An elevated removal ofUV-induced pyrimidine dimers from the transcribed compared to the nontranscribed strand of the D H F R gene in cultured CHO and human ceils was found by Mellon et al. (1987). The nontranscribed strand was repaired either not at all (CHO cells) or very slowly (normal human cells), compared with the very rapid and complete repair of the transcribed strand (Mellon et al. 1987). Similar results were reported by Smerdon and Thoma (1990) for the repair of UV damage on a plasmid transfected into yeast cells. The advantage to the dividing ceils of repairing more efficiently its transcribing compared with its nontranscribing strand and its implications in molecular evolution need to be shown. Indeed, it is the nontranscribed strand, presumably more mutated according to these studies (Mellon et al. 1987), that will produce, by replication, the transcribed strand in daughter cells. McGregor et al. (1991) used UV-irradiated synchronous cultures of xeroderma pigmentosum and normal diploid human fibroblasts to isolate 6-thioguanine-resistant mutants resulting from loss of function of the HPRT gene. Sequencing of the HPRT gene in 84 different mutants has shown that, although the transcribed strand contains 75% of the lesions (25% are formed in the nontranscribed strand), it is preferentially repaired in normal but not in xeroderma pigmentosum cells. Thus, the distribution of lesions is reversed. Cyclobutane dimers were found to be preferentially removed in E. coli from the transcribed over the nontranscribed strand in the lac Z (Mellon and Hanawalt 1989) and in the lac I genes (Koehler et al. 1991). A similar observation was made by Vileling et al. (1989, 1991) for the HPRT gene and by Venema et al. (1991) for the repair of ADA and D H F R genes in both xeroderma pigmentosum and normal human fibroblasts. This difference was thought to arise from both a preferential repair of the transcribed over the nontranscribed strand as well as from a higher fidelity of DNA replication of the nontranscribed strand (that will produce the transcribed strand in daughter cells) (Vrieling et al. 1991). Mechanistically, a difference in the repair rate between the two strands of the same gene might arise from the possibility that different types of DNA polymerases seem to be involved in their replication. The leading strand is replicated by DNA polymerase 6 (Stillman 1989), an enzyme containing an

164 Nuclear Matrix Proteins \

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Fig. 1. A schematic diagram showing how a protein might recognize a DNA lesion (A) and by virtue of its binding to the nuclear matrix or its interaction with protein components of the nuclear matrix might mediate the anchoring of a damaged DNA site to the matrix. Nuclear matrix is the principal site where DNA repair takes place (Mullenders et al. 1988). Attachment of damaged DNA to the nuclear matrix is suggested here to be the first, essential step for efficient repair. Xeroderma pigmentosum (XP) and Cockayne's syndrome (CS) cells that are unable to perform the incision step in the excision repair of UV damage may lack the protein factors that recognize UV-induced lesions on DNA and may thus be unable to anchor damaged sites to the matrix. According to this hypothesis, actively transcribed genes considered to be constitutively attached to the nuclear matrix would be repaired much more efficiently than inactive genes in XP and CS cells, a hypothesis supported by experimental evidence (Mullenders et al. 1984; Venema et al. 1990a,b).

165 associated 3' -~ 5' exonuclease proofreading activity (Bambara and Jessee 1991). On the other hand, the lagging strand seems to be replicated by DNA polymerase a (Hurwitz et al. 1990), which can be isolated free of exonucleolytic activity (Bambara and Jesee 1991). However, exonucleolytic proofreading occurs on both strands of SV40 DNA during its bidirectional replication by HeLa cell extracts; indeed, base substitution error rates were found to be similar during replication of the leading and lagging strands (Roberts et al. 1991). This finding argues against an error-prone 6 polymerase causing the increase in mutation rate in the transcribed compared to the nontranscribed strand (assuming that the origin of replication is located toward the 5' end of the gene and the transcribed strand is the leading strand during replication).

4. DNA Replication Errors and Proofreading Ability of DNA and RNA Polymerases During the replication of DNA, about one error is made every 106 nucleotides of newly made DNA; this is due to an iminotautomeric form of cytosine occurring at a frequency of one in one million that pairs with adenine rather than guanine and to a lactim form of thymine, similarly occurring in one in every million nucleotides that pairs with guanine rather than adenine as first predicted by Watson and Crick (1953). This gives about 3000 errors per cell (3 z 109 bp of DNA) per generation in human haploid cells, which is higher than the actual mutation rate; thanks to the proofreading activity of some DNA polymerases based on their 3' --, 5' exonuclease activity, such errors are repaired and the actual rate of fixed mutations is about one every 109-10 l° bp per cell duplication in E. coli (see Echols and Goodman 1991). Some clues indicate that GC-rich segments of DNA might be subject to more errors than AT-rich segments during replication (Chang 1973; see Loeb and Kunkel 1982; Modrich 1987). Considerable differences in the proofreading ability of RNA and DNA polymerases exist in nature. For example, the HIV reverse transcriptase has no proofreading activity and introduces one base-substitution error about every 2000 nucleotides (Preston et al. 1988). This could explain the rapid generation o f HIV variants evolving at a rate approximately one million times as great as that of eukaryotic DNA genomes (Hahn et al. 1986). The DNA-dependent RNA polymerase activity associated with calf t h y m u s D N A polymerase (primase) responsible for the synthesis of the RNA primer during replication is the least accurate polymerase known (Zhang and Grosse 1990). The high frequency of errors observed for RNA polymerases might be a consequence of the possibility that repair

mechanisms were not invented for the evolutionarily older RNA genomes. Reverse transcription of RNA molecules into DNA and their integration into the genome as retroviral-like transposable elements might be a means to speed up evolution (Cairns et al. 1988). Induction of reverse transcription by environmental stress has been observed in Drosophila (Strand and McDonald 1985) and in yeast (Rolfe et al. 1986). Those DNA polymerases from higher eukaryotes that have been tested in vitro show a much higher misincorporation rate than expected from spontaneous mutation rates in vivo (Loeb and Kunkel 1982). In the absence of environmental stress, variants in a population arise predominantly from replication errors (Luria and Delbrfick 1943; Wintersberger 1991). Is there a lower frequency of errors introduced during early replication of active genes compared to error frequency in the late-replicating nontranscribed part of the genome? Such a difference might arise, for example, by reactive oxygens and free radicals accumulating in the nucleus during replication, an intense metabolic activity during the S phase, and preferentially attacking the replicating DNA, presumably more vulnerable to mutagens.

5. Repair Pathways It is worthwhile to point here to the known complexity of the different repair pathways. Cells have invented a great number of repair pathways in order to cope with the different types of damage incurred by the environment and the cell metabolic processes. A significant number of cellular genes exist that may encode for repair enzymes. Excision repair is the more generalized repair pathway acting on diverse DNA lesions including those produced by UV irradiation and a broad class of chemicals (Pu et al. 1989; van Houten 1990). In E. coli six different proteins cooperate to carry out the excision repair of a segment of DNA. Uvr (A) and Uvr (B) form a complex that can act as an ATPdependent helicase and translocate along the DNA and arrest at DNA lesions. Uvr (C) protein then binds and incises the DNA 7 nucleotides upstream and 3 or 4 nucleotides downstream of the lesion. The 11- or 12-nucleotide strand segment containing the damage and the bound proteins are displaced by DNA polymerase I and the helicase activity of Uvr (D). Finally, the resulting gap is filled in by DNA polymerase I, and the nick is sealed by ligase II (see Grossman and Yeung 1990; Selby and Sancar 1990; van Houten 1990; Wood and Coverley 1991). A number of mammalian genes have been isolated that are involved in the excision-repair pathway. The general strategy leading to the isolation of

166 these genes involves transfection of repair mutant cell lines with normal genes and selection of genes that can confer mutagen resistance to mutant cells (see Hoeijmakers and Bootsma 1990; Wood and Coverley 1991). The XPAC gene thus isolated, complements the xeroderma pigmentosum A group and encodes for a zinc-finger protein (Tanaka et al. 1990). The ERCC-3 or XPBC gene encodes for a DNA helicase that is defective in patients suffering with xeroderma pigmentosum and Cockayne's syndrome (Weeda et al. 1990). The ERCC-2 gene exhibits high homology to the yeast Rad3 gene and encodes, like XPAC and ERCC-3 genes, for a protein that interacts with DNA (Weber et al. 1990). Excision repair in mammalian cells starts with an endonuclease cut about 8 bp upstream from the lesion. The incision is followed by the removal of a short (13-15 nucleotides) or a long stretch of deoxynucleotides containing the lesion (Harwood et al. 1991; Thomas et al. 1991). This step is followed by repair synthesis in the resultant gap using the undamaged complementary strand as template, and finally repair is completed by ligation of the repair patch to the parental DNA strand with ligase II (reviewed by Cleaver 1978; Lindahl 1982). A variety of enzymes are specialized for the repair of small modifications to single damaged bases (Lindahl 1990). Spontaneous depurinations in DNA seem to occur at a frequency of about 104 purines per human cell per day at 37°C. Specific enzymes (invertases) may recognize depurinated sites and may replace the proper purine directly without disrupting the phosphodiester bond (Deutsch and Linn 1979; Livneh et al. 1979). Most frequently, however, depurinated sites in DNA are repaired via excision repair (Lindahl 1982). O-methylation may be repaired by the direct removal of the methyl group catalyzed by a specific O6-methylguanine-DNA methyltransferase (Pegg et al. 1983; Dolan et al. 1988). Individuals among humans may display different levels in this enzyme activity (Oesch et al. 1987; Souliotis et al. 1989), and some deficiencies in this enzyme may increase cancer risk (Montesano 1981). Both the sites of formation and repair of O6-methylguanine are nonrandomly distributed on DNA (Topal et al. 1986; Topal 1988). The presence of Z-DNA seems to preclude the repair of O6-methylguanine (Boiteux et al. 1985). A 3' phosphodiesterase is required for the repair of single-strand breaks on the D N A produced by ionizing radiation or bleomycin, producing D N A fragments with 3' phosphate groups. Indeed, fill-in of the gaps by D N A polymerases during repair requires a free 3' hydroxyl group. A 3' phosphodiesterase was purified from calf thymus, and its cDNA from bovine libraries was sequenced (Robson et al.

1991). A similar Drosophila enzyme cDNA has been isolated by Kelley et al. (1989). DNA glycosylases catalyze the hydrolysis of damaged base-sugar bonds to release deaminated, alkylated or oxidized bases (reviewed by Sakumi and Sekiguchi 1990) as well as mismatched G . T base pairs (Wiebauer and Jiricny 1990) and G . A base pairs (Au et al. 1989). Specific glycosylases like the 3-methyladenine DNA glycosylase is able to remove a mispaired base without cleavage of the DNA strand (Helland et al. 1987). A thymine-specific glycosylase in human cell nuclei removes the thymine in a G . T mispair, generating an apyrimidinic site; the single nucleotide gap is then filled in by DNA polymerase/3 (Wiebauer and Jiricny 1990). The generation of G. T mispairs is especially important in the present connection. Spontaneous deaminations, occurring at high frequency in DNA, convert cytosine to uracil, adenine to hypoxanthine, and guanine to xanthine. The G. U mispair can be repaired by uracil-DNA glycosylases (Lindahl et al. 1977; Frederico et al. 1990). An evolutionary conservation between the human and E. coli uracil-DNA glycosylase has been observed. The hypoxanthine arising from deamination of adenine can be released by hypoxanthine-DNA glycosylase purified from calf thymus (Dianov and Lindahl 1991). Are there chromatin fractions more accessible to these enzymes than others? Although no studies are available addressing this issue, we might expect active chromatin fractions to be more accessible than transcriptionally inactive chromatin to such repair enzymes. The great majority of the G. T mismatches arise by deamination of 5-methylcytosine in DNA yielding thymine; thus a G-meC pair is converted into G" T. A greater number of G" T mismatches would be expected to arise in inactive genes and in the inactive X-chromosome containing a higher level of methylated cytosines (Cedar 1988). Therefore, cells having low activities in the thymine-specific glycosylase will be unable to correct the G-T mismatches and would generate a G. C pair in the one and an A-T pair in the other daughter cell during replication. Thus, it is predicted that a higher mutation rate of this specific type might be present in the inactive part of the genome.

6. Low Repair Activity, Recombination Events, and Sequence Hypervariability According to the neutral theory, the frequency of alleles of a gene or DNA sequence in a genome at any given time during molecular evolution represents a temporary frame from an ongoing dynamic process; alleles in polymorphic loci are either on

167 their way to fixation or are about to become extinct (Li and Graur 1991). In cage populations of Drosophila, a slower rate of loss of polymorphisms was found by Powell (I 971) when the environment was diversified than when it was constant. Here we point to some extreme examples ofpolymorphic sequences and summarize the mechanisms that may generate polymorphism. Specific loci in differentiated somatic cells such as the MHC locus (Klein 1978, 1979), the hypervariable segment in the immunoglobin genes (Moynet et al. 1985), and the T-cell receptor genes (Barth et al. 1985) are polymorphic. The high variation within the hypervariable region of the immunoglobulin heavy- and light-chain genes is mainly generated by modulation of the precise recombination sites during immunodifferentiation (Sakano et al. 1979; Seidman et al. 1979) but also by mutation events. Bacterial lipopolysaccharides, known to activate transcription of the K immunoglobulin gene, also activate these recombination events (Schlissel and Baltimore 1989). This observation parallels that of Powell (1971) for Drosophila (see above). Immunoglobulin genes show substantial changes in their chromosomal organization and in the arrangement of their gene segments during evolution; however, their basic mechanisms of recombination are similar among vertebrates (Hinds and Litman 1986). Specific recombinases (Matsunami et al. 1989; Schatz et al. 1989), in addition to a deficiency in DNA repair mechanisms specifically operating in switch regions of immunoglobulin genes (Dunnick and Stavnezer 1990), seem to be responsible for the polymorphism in these loci. The mutations that can be found in some immunoglobulin heavy-chain switch regions after recombination appear to arise only on one strand of DNA, probably by error-prone synthesis of that one strand (Dunnick and Stavnezer 1990). The switch region of immunoglobulin heavy-chain genes is composed of simple tandem repeats of the 5-bp unit sequences GAGCT and G G G G T (Nikaido et al. 1981) that are G rich and might be expected to acquire lesions more easily (section I, part 1). Analysis of DNA repair in the immunoglobulin genes has shown that lymphocytes active in immunoglobulin gene rearrangements were less efficient than granulocytes in removing pyrimidine dimers from the r and # gene domains (Bianchi et al. 1990). Regions of poor repair seem to be also regions displaying a high frequency of recombination (Bianchi et al. 1991). Such a locus exhibiting low repair activity and enhanced break points in 50% of human Burkitt lymphomas and 70% of murine plasmacytomas is exon 1 or intron 1 of the c-myc gene (Bianchi et al. 1991). Tandemly repeated short DNA elements called

hypervariable minisatellites (Jeffreys et al. 1985; Wyman et al. 1985) are proximal to recombination hot spots in the mouse MHC (Steinmetz et al. 1986), to chiasmata in meiotic h u m a n c h r o m o s o m e s (Chandley and Mitchell 1988), and to the known recombination hot spot Chi in bacteriophage X (Smith et al. 1981). Hypervariable minisatellites seem to cause a 13-fold stimulation in intermolecular homologous recombination in the human genome (Wahls et al. 1990). Specific minisatellitebinding proteins might be responsible for the enhanced recombination frequency in these sequences (Wahls et al. 1991). Thus, additional mechanisms other than the preferential repair of different chromatin domains may contribute to genome fluidity. Alu sequences, repeated about 900,000 times in the human genome, arose by retroposition at different time intervals of human evolution (Britten et al. 1988; Jurka and Smith 1988). Alu sequences seem to be involved in recombination events, often shuffling, amplifying, or deleting gene segments and resulting in human hereditary diseases (Calabretta et al. 1982; Lehrman et al. 1987; Hu et al. 1991). Junctions at tandem duplications ofdystrophin gene segments were found to involve Alu and topoisomerase I and II consensus cleavage sequences (Hu et al. 1991). Source Alu sequences (10-20 Alu sequences in total per genome) are conserved in evolution (Britten et al. 1989). However, the vast majority of Alu sequences might evolve at a rate very close to what is considered a neutral rate. It remains to be shown whether or not a limited amount of selection in nonsource Alu genes might take place (Zuckerkandl et al. 1989). it would be interesting to compare repair rates between source and nonsource Alu genes. In summary, DNA polymorphism in a given DNA sequence may be generated by recombination, by the enhanced vulnerability of a specific sequence to damage, by deficiency in DNA repair specifically operating at this sequence, and by either neutral drift or natural selection. How specific repair mechanisms might be absent from a specific DNA sequence but not from the majority of DNA sequences within the same nucleus and how higher-order chromatin structures or different segments ofchromatin loops intervene to exclude repair needs to be investigated.

7. Chromatin Structure, Nuclear Matrix, and DNA Repair Repair mechanisms require changes in chromatin structure believed to be mediated via nuclear protein poly(ADP-ribosyl)ation (reviewed by Boulikas 1991, 1992). High repair activities are directly related to high poly(ADP-ribosyl)ating activities, and

168 poly(ADP-ribose) polymerase is a repair enzyme. This enzyme is unique in that it is activated by DNA strand breaks, and strand breaks stimulate histone poly(ADP-ribosyl)ation (Boulikas 1988, 1989, 1990). We do not know the exact changes in nucleosome and higher-order chromatin structure caused by histone poly(ADP-ribosyl)ation; however, poly(ADP-ribose) molecules might open higher-order chromatin structures, unfold individual nucleosomes, or temporarily remove core histone octamers from DNA at chromatin sites undergoing repair (Boulikas 1992). Poly(ADP-ribosyl)ation might regulate the activity of enzymes that might be involved in repair; such enzymes, including topoisomerases I and II, DNA polymerases, ligase II, and endonucleases, might become inactive after their poly(ADPribosyl)ation and be reactivated after degradation of poly(ADP-ribose) by glycohydrolase (see Boulikas 1991). The nuclear matrix is an important structure where transcription, replication, repair, and recombination presumably take place (for a review see Boulikas 1992). Nuclear matrix contains topoisomerase II as a structural component (Berrios et al. 1985) and includes a small percentage of the nuclear DNA that seems to be as low as 5% of the total DNA. Nuclear matrix DNA is AT rich, contains sequence motifs that bend easily (Homberger 1989), and is enriched in origins of replication (Rao et al. 1990; Razin et al. 1991; Boulikas and Kong, unpublished). However, the possible enrichment of origins of replication in cruciform structures (Zannis-Hadjopoulos et al. 1988) may render them refractory to repair enzymes (Topal 1988). Low repair of the origin of replication of an artificial autonomously replicating plasmid after its transfection in yeast cells has been noted (Smerdon and Thoma 1990). Thus, a fraction of matrix DNA comprising origins of replication may display a high mutation rate. We do not know whether mammalian origins of replication are polymorphic due to our inability to efficiently isolate and study mammalian origins of replication (Russev and Vassilev 1982; ZannisHadjopoulos et al. 1985). This study predicts that a fraction of the nuclear matrix DNA, including sequences functioning as origins of replication, might be polymorphic. In spite of our ignorance of evolutionary conservation in mammalian origins of replication, origins in general appear to be hypervariable. The mitochondrial origin of replication of 400 bp contains most of the hypervariability found in the mitochondrial genome (Upholt and Dawid 1977; Sekiya et al. 1980; Anderson et al. 1981 ). The origin of replication of herpes simplex virus type 1 consists of a perfect 144-bp palindrome that is prone to deletion (Weller et al. 1985). Similarly, the origin

of replication ofE. coli seems to be involved in the process by which 7-ray-induced deletions take place (Raha and Hutchinson 1991). Another DNA component of the nuclear matrix has been highly conserved in evolution in order to undertake its structural role in organizing chromatin into domains (Boulikas et al., unpublished).

III. Preferential Damage and Repair in Germ-Cell Lineages The preferential damage and repair of different chromatin domains in germ-cell lineages and, as a result, the nonrandom introduction of mutations with respect to active, inactive, and nontranscribed DNA sequences in germ cells are of special interest in molecular evolution. Indeed, the mutations to be transmitted to progeny are those introduced into germ cells. During spermatogenesis in the testis, a complex series of events takes place in which a terminally differentiated cell, the spermatozoon, is produced from a stem cell (see Clermont 1972; Fawcett 1975). Although testis tissue is richer in poly(ADP-ribose) polymerase m R N A than most mouse tissues (Ogura et al. 1990) and thus is expected to be high in repair activities (see Boulikas 1991, 1992), mature spermatocytes show almost no poly(ADP-ribosyl)ation (Corominas and Mezquita 1985). This observation is compatible with the absence of DNA repair (Chandley and Kofman-Alfaro 1971; Sega 1974) and of transcriptional activity (Kierszenbaum and Tres 1975) in mature spermatozoa. Testis chromatin appears to possess a number of histone variants not found so far in other organs. Such variants include a testis-specific H 1 or H 1t as well as testis-specific H2A, H2B, and H3 variants (e.g., Shires et al. 1975; Seyedin et al. 1981; Meistrich et al. 1985; Cole et al. 1986; Grimes et al. 1987; Kim et al. 1987). The role of germ-cell-specific histones in chromatin structure during spermatogenesis is not clear. Temporal differences of accumulation of testis historic variants in spermatogonia and spermatocytes indeed point to a specific role for each variant in chromatin structure and function. DNA damage also has been shown to be reduced during spermatogenesis. During this process, the DNA in spermatocytes is completely repackaged by small, arginine-rich proteins the protamines 1 and 2 (Balhorn 1989). DNA is neutralized, and the D N A protamine complex is in a maximally condensed, biochemically inert state. The high affinity of protamines for the minor groove of DNA (Herskovits and Brahms 1976; Suau and Subirana 1977) and the marked insolubility that characterize the struc-

169 ture of the sperm chromatin render the DNA inaccessible to intercalating agents known to bind DNA in its minor groove such as actinomycin D (Darzynkiewicz et al. 1969; Balhorn et al. 1985). In addition, a mutagenic DNA alkylating agent, benzpyrene, which alkylates the N-2 position of guanine, is unable to damage sperm DNA (see Balhorn 1989). In the early pronuclear stage of the fertilized egg, the protamine surrounding DNA of sperm is discarded and the typical nucleosomal structure is reestablished. This chromatin remodeling renders pronuclear sperm DNA more vulnerable to x-ray damage compared to the mature sperm (Matsuda et al. 1989b). These lines of evidence strongly suggest that DNA in mature sperm might be insensitive to most mutagens. However, the stage of formation of mature sperm (spermiogenesis) represents only a final stage in spermatogenesis. For example, in the mouse, the meiosis that precedes spermiogenesis lasts 11-12 days, compared to 14 days required for the spermiogenesis stage itself(Hecht 1989). In humans, the entire process ofspermatogenesis lasts 64 days compared to about 26 days in the mouse (see Clermont 1972). Spermiogenesis has been divided by morphological criteria into 16 steps, complete genome silencing by replacement of histones by protamines is seen only in the late spermiogenesis steps. For example, repair activities in the germ cells of male mice occur from leptotene through midspermatid stages (Sega 1990). Thus, a general protection against mutations attributable to DNA packaging by protamines would apply to the latest stages of spermatogenesis when repair is absent. Someone could ask whether or not the higher mutation rate for the nontranscribed part of the genome predicted for somatic cells (see section IV, part 2) applies to the germ cells as well. From the high repair activity of mammalian germ cells (Sega 1990), it is predicted that repair of active genes in testis is more efficient than repair of the nontranscribed part of the genome. Thus, male germ-cell lineages are expected to follow the same pattern as somatic cells, i.e., to display a higher mutation rate in the nontranscribed part of the genome. It is not clear why ENU-induced mutations on germ cells are confined to a single DNA strand (Favor 1990). In addition, we do not understand why the point mutation induced in the a-globin gene in germ cells by ENU was an A . T ~ G. C transition (Peters et al. 1990). This contrasts with data from cellular systems showing that ENU causes G -~ A transitions (Loechler et al. 1984; Richardson et al. 1987; section I, part 3). It should be emphasized that roughly 15% of the DNA in mature human sperm is packaged into typical nucleosomes (Tanphaichitr et al. 1978; Gusse

et al. 1986; Gatewood et al. 1987, 1990). This part of the sperm genome that might facilitate the programming of genes that will be active in early development (Gatewood et al. 1990) is thus expected to be more vulnerable to mutagenic agents. However, mouse oocytes appear to have a normal capacity to repair damage on the sperm DNA upon fertilization (Generoso et al. 1979; Brandriff and Pedersen 1981; Bennett and Pedersen 1984; Matsuda and Tobari 1989; Matsuda et al. 1989c; Cattanach et al. 1990). Unfertilized mammalian oocytes appear to have high repair capacities (Guli and Smyth 1989). A decrease in repair activities at the time of meiotic maturation in mouse oocytes has been noted (Guli and Smyth 1988), but fully mature oocytes maintain a high repair capacity in contrast to the mature sperm (Pedersen and Brandriff 1980). Upon fertilization, the repair capacity of eggs remains high (Generoso 1980; Brandriff and Pedersen 1981; Bennett and Pedersen 1984; Matsuda et al. 1989a). It is reasonable to postulate that in their repair capacities mammalian oocytes, fertilized eggs, and early embryos resemble actively proliferating somatic cells in culture. Thus, the preferential repair of active genes compared with the transcriptionally inactive part of the genome found for cells in culture might apply to male germ cells in premeiotic stages of spermatogenesis, oocytes, fertilized eggs, and early-stage embryos.

IV. Evolution

1. Damage Tends to Increase the AT-Richness of Sequences Most of the damaging agents (MNNG, MNU, UV, BrdU, 3-methyl cholanthrene, aflatoxin, HO-AAF), including the C -~ T transversions arising by deamination of 5-methyl-C and its conversion into T (Pfeifer et al. 1991), primarily occurring in transcriptionally inactive DNA sequences, tend to diminish the GC content of genomes (see section I, part 3). Conversely, if the action of these genotoxic agents can be generalized, one would infer that evolution toward higher GC content including evolution in isochores (Bernardi et al. 1985), being contrary to most expected environmentally induced effects, is likely to be attributable to natural selection. This contention agrees with the proposal made by Ticher and Graur (1989) stating that synonymous mutations giving rise to A and T are selected against and result in diminished rates of synonymous substitution in protein-coding regions in comparison to the rate of substitution in pseudogenes. Their proposal was based on a systematic study of the rates

170 of synonymous substitutions in 42 protein-coding gene pairs from rat and human. Thus, selection and mutation seem to work in opposite directions within coding regions of genes, with mutation resulting in AT richness and selection removing it. The much higher levels of G ~ A and C --, T transitions caused by UV, DNA alkylating agents, and several other mutagens predict that sequences not bound by selective pressures tend to become more AT rich due to the presence of such mutagens in the environment. Examination of DNA sequences that are under no selective constraints, as, for example, pseudogenes, has indeed shown an increase in their AT content during evolution (Gojobori et al. 1982; Li et al. 1984; Li and Graur 1991). In several cases, however, gene-coding sequences might display a higher G ~ A and G -~ T substitution frequency during evolution compared, for example, with T ~ C or T --, G and thus evolve toward a slight AT richness. This has been found, for example, in the cytochrome c genes (Zuckerkandl et al. 1971) and in the 3-globin genes in primates (Bulmer 1991). Sequence has an important effect on local rate of damage undergone by DNA. Because base composition presumably directs the chances of occurrence of sequence motifs, it can be anticipated that rates of DNA damage will be different in different isochores (Bernardi et al. 1985). GC-rich isochores are predicted to be attacked at higher rates than ATrich isochores. With the advent of homeothermy during vertebrate evolution, there has been a trend toward the formation of a larger component of GC-rich isochores; the compositional changes that occurred during the evolution from cold- to warmblooded vertebrates were brought about by directional point mutations (Bernardi and Bemardi 1991). It remains to be investigated what roles unrepaired DNA damage, error in DNA replication, and natural selection have each played in this process. One important question is what types of DNAdamaging agents have been the major mutationcausing agents during the last one billion years or so of molecular evolution. UV radiation, 3, rays, and specific types of chemicals such as reactive oxygens produced during metabolism in the cell have certainly contributed to DNA strand break-induced rearrangements, duplications, and deletions. Specific chemicals or damaging agents may have been abundant during specific periods of time and in certain microenvironments such as chemicals in areas of volcanic eruptions and 3' rays in areas containing natural radioactivity. The average direction and rate of base injuries should vary during evolution if the predominant genotoxic agents of environmental origin change over time. For instance, before the earth's ozone layer

was formed, average trends in compositional change ofgenomes are expected to have been quantitatively different from what they were afterward. The rates, if not the balance of the trends themselves, should be a function of the proportion of UV light reaching the surface of the earth. Such variations are of biological import only to the extent to which they affect the evolution of functions in organisms.

2. Evolutionary Consequences of the Preferential Damage and Repair of Active over Inactive Genes DAMAGE, REPAIR, AND THE NEUTRAL THEORY OF EVOLUTION It appears that active genes are both damaged and repaired at higher rates than transcriptionally inert regions of the genome. Some exceptions apply, as, for example, the spontaneous deamination of 5-methylcytosine into thymine (Wiebauer and Jiricny 1990) occurring more frequently within transcriptionally inactive sequences that are known to be enriched in methylated DNA (Cedar 1988). The balance between damage and repair may not have remained constant for a given gene during molecular evolution. A higher mutation rate in the transcriptionally inactive compared with the transcriptionally active part of the genome has been found during molecular evolution; for example, pseudogenes display a higher mutation rate than normal genes (e.g., Li and Graur 1991). Are there any differences in the mutation rate between active and inactive parts of the genome in somatic versus germ cells? Are there differences in repair rates between active and inactive genes on one hand and noncoding intergenic DNA sequences on the other hand? These issues in spite of their importance in molecular evolution have not been approached experimentally. According to the traditional view of molecular evolution, the rate of point mutation is uniform over the genome of an organism, and variation in the rate of nucleotide substitution among DNA loci reflects differential selective constraints (Kimura 1983; see, however, Wolfe et al. 1989; Li and Graur 1991). From the present study, providing strong evidence that there is a preference of damage of active over inactive chromatin fractions in proliferating cells and from the well-established preference of repair of active over inactive DNA sequences (e.g., Hanawalt 1989), it is predicted that the rates of nucleotide substitution arising from unrepaired DNA may be higher within DNA sequences harbored in transcriptionally inactive chromatin structures. This conclusion is based on the observation that repair wins over damage in actively proliferating ceils (see section II). The base composition of some short DNA sequences within genes that renders them 2-100-fold

171

more vulnerable to UV (e.g., Brash et al. 1987; Pfeifer et al. 1991; see section I, part 2) and to DNA alkylating agents (Mattes et al. 1986, 1988; Dolan et al. 1988) might explain the accelerated evolution observed in some Drosophila chorion genes (Martinez-Cruzado 1990) or in some mammalian genes (see Ohta 1991). Whether genes or gene segments displaying the highest rate of evolution coincide with a high vulnerability to mutagens or low repair need to be examined. Because, according to the neutral theory, the rate of substitution would be equal to the rate of neutral mutations, gene segments with different functions would display evolutionary rates inversely proportional to the degree of selective constraints operating on them (Wilson et al. 1977). It would be exciting to find a correlation between the degree of selective constraints that apply on a gene segment and its rate of damage and repair. In a few cases, a high rate of amino acid replacement in a functional domain of a protein might imply that positive Darwinian selection is occurring as has been shown in the stomach-specific lysozyme gene in the langur (Stewart et al. 1987) and in the variable region of immunoglobulin genes (Tanaka and Nei 1989). The variable region of immunoglobulin genes in active lymphocytes seems to be repaired poorly (Bianchi et al. 1990, 1991) and thus supports the contention that high rates of damage or low rates of repair would speed up molecular evolution at specific DNA sequences. The preference of several damaging agents for attacking active rather than inactive genes (e.g., Ramanathan et al. 1976; Walker et al. 1979; KohwiShigematsu et al. 1983; Ryan et al. 1986) may be due to unusual DNA structures in the regulatory regions of active genes induced by torsional strain generated during transcription, to the absence of nucleosomes in the 5' flanking regulatory regions of active genes, to their "open" nucleosomes and to their uncondensed chromatin structure. DNA repair mechanisms in normal proliferating mammalian cells can overcome the preferential damage of actively transcribed sequences and even distinguish between the transcribing and nontranscribing strand of the same gene and repair the transcribing strand more efficiently. This will result in a lower mutation rate in the active over inactive genes and in the transcribed t h a n in the nontranscribed strand (McGregor et al. 1991). However, quiescent cells that are known to be low in repair activities (e.g., Liu et al. 1983; Greer and Kaplan 1986) might be expected, according to our hypothesis, to have a higher mutation rate in the active part of their genome due to its vulnerability to damage. The occurrence of a higher mutation rate in the active than in the inactive part of the genome was first suggested by Zuckerkandl (1985).

LIFE SPAN AND REPAIR CAPACITY AMONG ORGANISMS

Organisms seem to differ substantially in their repair efficiencies as well as in the overall mutation rates. For example, the rates of repair of thymidine dimers are much higher in human than in rodent cells in culture (Ganesan et al. 1983). There is a striking correlation between DNA repair capacities and life span among primates (Hall et al. 1984). In view of the high degree of similarity in protein and DNA structure among primates, a maximum life span difference of more than 20-fold among primates (Cutler 1975) might arise from a correlation between repair capacity and longevity. Indeed, species displaying the longest life span within a group of closely related species such as primates are those with the highest ability in their cells to perform DNA repair efficiently (Hall et al. 1984). The correlation between life span and DNA repair capacity exists among mammalian species of different families (e.g., Hart and Setlow 1974; Hart et al. 1979; Hart and Modak 1980). These lines of evidence demonstrate the importance of DNA repair in longevity. An alternative hypothesis, however, is that short-lived species have a shorter generation time and a higher number of germ-line DNA replications per year arising from errors during replication (Kohne 1970). This factor may contribute to the higher substitution rates in rodents than in humans in addition to the known higher efficiency of human compared with rodent cells to repair DNA (Ganesan et al. 1983; Mellon et al. 1987). MUTATION RATES IN ACTIVE AND INACTIVE GENES

A systematic study by Wolfe et al. (1989) has shown a significant variation in mutation rate among regions of the mammalian genome. The rate of silent substitution was found to vary among genes and was attributed to systematic differences in the rate and pattern of mutation over regions of the genome (Wolfe et al. 1989). The present study predicts that the probability of the initial appearance of a lesion within a specific gene would depend upon its higher-order chromatin structure, DNA base composition, transcriptional activity, cell type, and the nature and concentration of mutagen. The rate of repair of a particular lesion would depend upon the type of lesion, the repair activity in the cell type, the transcriptional activity and higher-order chromatin structure of the locus where the lesion has occurred, the proliferating state of the cells, and the type of organism. Active genes are expected to be damaged at higher rates but also to be repaired at higher rates. Assuming that repair mechanisms similar to those operating in somatic cells in culture apply to germ cells, and because in

172 proliferating cells repair wins over damage (e.g., Vrieling et al. 1989, 1991; McGregor et al. 1991; Koehler et al. 1991), active genes might have a lower mutation frequency during evolution than nontranscribed DNA. Active genes may include the repertoire o f genes that are active in germ cells. Genes containing potential Z-DNA, H-DNA, or cruciform structures in their regulatory regions that are refractory to repair enzymes (Topal 1988) might have a higher mutation rate than the genome overall. O N THE MOLECULAR CLOCK HYPOTHESIS

Zuckerkandl and Pauling (1965) have formulated the molecular clock hypothesis, stating that the rate o f evolutionary divergence of a given genetic unit whose function has been fixed long ago remains constant over time (see also Zuckerkandl 1976). Because the rate of damage of a given D N A sequence greatly depends upon its base composition and may be as much as two orders of magnitude higher than the rate o f damage of surrounding sequences (see section I, part 2), some specific genes as well as specific sites within genes will be more prone to the introduction o f premutagenic lesions. Thus, repair and neutral drift will determine the rate of fixation o f mutations in these particular genes, except to the extent to which natural selection is operating. The rate o f evolution of a particular gene or gene segment would depend (l) upon its vulnerability to mutagens determined by its DNA base composition and some other factors (see section I) that are almost constant for a particular gene over significant periods of evolutionary time; (2) upon its rate of repair that will be determined by its chromatin structure in the germcell lineages and some other factors described in section II that again appear to be constant for a given gene; and (3) upon natural selection. This study gives support to the molecular clock hypothesis when applied to one gene within a group of related species. Because there are differences in the rate o f repair for the same gene due to differences in the overall repair efficiencies observed among species, the rate of evolutionary divergence o f a given gene among distant organisms may not be constant. Thus, we predict that the rate of amino acid substitutions of a given protein among a group of related species that have similar gene sequences and repair efficiency, such as primates, will be constant over geological time, in agreement with Zuckerkandl and Pauling ( 1965), Kimura (1969), and Dickerson (1971), but will be variable among distant species that differ in gene organization and repair efficiency, in agreement with G o o d m a n (1981), Kunisawa et al. (1987), and H or i m ot o et al. (1990). This study predicts that the evolutionary rate will vary between genes, in agreement with Dickerson (1971), Ticher and Graur (1989), and others, segments of a gene (Jones and Kafatos 1982; Thackeray and Kyriacou

1990; see Wilson et al. 1977) and synonymous sites of the same gene (Riley 1989), not only as a result of differences in selective constraints that apply to each genetic unit, particular gene segments or gene regulatory regions, but also as a result of differences in the rate of damage minus rate of repair among different segments of chromatin DNA. Acknowledgments. Very special thanks and gratitude to E. Zuckerkandl for his encouragement, excellent discussions, and valuable input on the manuscript, G. Bernardi and E. Trifonov for their critical comments, to F. Clairvoyant for his kindness and patience in preparing the manuscript, to R. Babcock for literature searches and for critically reading the manuscript, and to J. Walichiewicz for computer graphics.

References Almer A, Rudolph H, Hinnen A, H6rz W (1986) Removal of positioned nucleosomes from the yeast PHO5 promoter upon PHO5 induction releases additional upstream activating DNA elements. EMBO J 5:2689-2691 Anderson S, Bankier AT, Barrell BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH, Staden R, Young IG (1981) Sequence and organization of the h u m a n mitochondrial genome. Nature 290:457-465 Armstrong JD, Kunz BA (1990) Site and strand specificity of UVB mutagenesis in the SUP4-o gene of yeast. Proc Natl Acad Sci USA 87:9005-9009 Au KG, Clarck S, Miller JH, Modrich P (1989) Escherichia coli rout Y gene encodes an adenine glycosylase active on G - A mispairs. Proc Natl Acad Sci USA 86:8877-8881 Balhorn R (1989) Mammalian protamines: structure and molecular interactions. In: Adolph KW (ed) Molecular biology of chromosome function. Springer-Verlag, New York, pp 366395 Balhorn R, Kellaris K, Corzett M, Clancy C (1985) 7-Aminoactinomycin D binding and the final stages of sperm chromatin processing in the mouse. Gamete Res 12:411-422 BambaraRA, JesseeCB (1991) PropertiesofDNApoylmerases 6 and ~, and their roles in eukaryotic D N A replication. Biochim Biophys Acta 1088:11-24 Barbacid M (1987) Ras genes. Annu Rev Biochem 56:779-827 Barth RK, Kim BS, Lan NC, Hunkapiller T, Sobieck N, Winoto A, Gershenfeld H, Okada C, Hansburg D, Weissman IL, Hood L (1985) The murine T-cell receptor uses a limited repertoire of expressed V~ gene segments. Nature 316:517-523 Beecham EJ, Mushinski JF, Shacter E, Potter M, Bohr VA (1991) DNA repair in the c-myc proto-oncogene locus: possible involvement in susceptibility or resistance to plasmacytoma induction in BALB/c mice. Mol Cell Biol 11:3095-3104 Belinsky SA, Dolan ME, White CM, Maronpot RR, Pegg AE, Anderson M W (1988) Cell specific differences in O6-meth ylguanine-DNA methyltransferase activity and removal of O6-methylguanine in rat pulmonary cells. Carcinogenesis 9: 2053-2058 Bennett J, Pedersen RA (1984) Early mouse embryos exhibit strain variation in radiation-induced sister-chromatid exchange: relationship with DNA repair. Murat Res 126:153157 Beranek DT, White GL, Heflich RH, Beland FA (1982) Aminofluorene-DNA adduct formation in Salmonella typhimurium exposed to the carcinogen N-hydroxy-2-acetylaminofluorene. Proc Natl Acad Sci USA 79:5175-5178 Bernardi G, Bernardi G (1991) Compositional properties of

173 nuclear genes from cold-blooded vertebrates. J Mol Evol 33: 57-67 Bernardi G, Olofsson B, Filipski J, Zerial M, Salinas J, Cuny G, Meunier-Rotival M, Rodier F (1985) The mosaic genome of warm-blooded vertebrates. Science 228:953-958 Bernardi G, Mouchiroud D, Gautier C, Bernardi G (1988) Compositional patterns in vertebrate genomes: conservation and change in evolution. J Mol Evol 28:7-18 Berrios M, OsheroffN, Fisher PA (1985) In situ localization of D N A topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc Natl Acad Sci USA 82:4142-4146 Bianchi MS, Bianchi NO, de la Chapelle A (1990) Assessment by Southern blot analysis of UV-induced damage and repair in h u m a n immunoglobulin genes. Mutat Res 232:89-97 Bianchi NO, Bianchi MS, Alitalo K, de la Chapelle A (1991) U V damage and repair in the domain of the h u m a n c-myc oncogene. DNA Cell Biol 10:125-132 BirnboimHC (1978) Spacing ofpolypyrimidine regions in mouse D N A as determined by poly(adenylate, guanylate) binding. J Mol Biol 121:541-559 Bodell WJ (1977) Non-uniform distribution of DNA repair in chromatin after treatment with methyl-methane sulfonate. Nucleic Acids Res 4:2619-2628 Bohr VA, Smith CA, Okumoto DS, Hanawalt PC (1985) DNA repair in an active gene: removal of pyrimidine dimers from the D H F R gene of CHO cells is much more efficient than in the genome overall. Cell 40:359-369 Bohr VA, Okumoto DS, Ho L, Hanawalt PC (1986) Characterization of a D N A repair domain containing the dihydrofolate reductase gene in Chinese hamster ovary cells. J Biol Chem 261:16666-16672 Bohr VA, Phillips DH, Hanawalt PC (1987) Heterogenous DNA damage and repair in the mammalian genome. Cancer Res 47:6426-6436 Boiteux S, deOliveira RC, Laval J (1985) The Escherichia coli O6-methylguanine-DNA methyltransferase does not repair promutagenic O6-methylguanine residues when present in Z-DNA. J Biol Chem 260:8711-8715 Boles TC, Hogan ME (1986) High-resolution mapping of carcinogen binding sites on DNA. Biochemistry 25:3039-3043 Bolshoy A, McNamara P, Harrington RE, Trifonov EN (1991) Curved D N A without A-A: experimental estimation of all 16 D N A wedge angles. Proc Natl Aead Sci USA 88:2312-2316 BoulikasT (1987a) N u e l e a r e n v e l o p e a n d c h r o m a t i n s t r n c t u r e . Int Rev Cytol Suppl 17:493-571 BoulikasT (1987b) Functions ofchromatin and the expression of genes. Int Rev Cytol Suppl 17:599-684 Boulikas T (1988) At least 60 ADP-ribosylated histones are present in nuclei from dimethylsulfate-treated and untreated cells. EMBO J 7:57-67 Boulikas T (1989) DNA strand breaks alter histone ADP-ribosylation. Proc Natl Acad Sci USA 86:3499-3503 Boulikas T (1990) Poly(ADP-ribosyl)ated histones in chromatin replication. J Biol Chem 265:14638-14647 Boulikas T (1991) Relation between carcinogenesis, chromatin structure and poly(ADP-ribosyl)ation. Anticancer Res 11:489527 Boulikas T (1992) Poly(ADP-ribosyl)ation, repair, chromatin and cancer (review). Curr Perspect Mol Cell Oncol 1:1-109 Brandriff B, Pedersen RA (1981) Repair of the ultraviolet-irradiated male genome in fertilized mouse eggs. Science 211: 1431-1433 Brash DE (1988) U V mutagenic photoproducts in Escherichia coli and h u m a n cells: a molecular genetics perspective on h u m a n skin cancer. Photochem Photobiol 48:59-66 Brash DE, Haseltine WA (1982) UV-induced mutation hotspots occur at D N A damage hotspots. Nature 298:189-192 Brash DE, Seetharam S, Kraemer KH, Seidman MM, Bredberg

A (1987) Photoproduct frequency is not the major determinant of U V base substitution hot spots or cold spots in h u m a n cells. Proc Natl Acad Sci USA 84:3782-3786 BreimerLH (1988) Ionizingradiation-inducedmutagenesis. Br J Cancer 57:6-18 Britten R J, Baron WF, Stout DB, Davidson EH (1988) Sources and evolution of h u m a n Alu repeated sequences. Proc Natl Acad Sci USA 85:4770-4774 Britten RJ, Stout DB, Davidson EH (1989) The current source of h u m a n Alu retroposons is a conserved gene shared with Old World monkey. Proc Natl Acad Sci USA 86:3718-3722 Brown K, Buchmann A, Balmain A (1990) Carcinogen-induced mutations in the mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression. Proc Natl Acad Sci USA 87:538-542 Bulmer M (1991) Strand symmetry of mutation rates in the fl-globin region. J Mol Evol 33:305-310 Cairns J, Overbaugh J, Miller S (1988) The origin of mutants. Nature 335:142-145 Calabretta B, Robberson DL, Barrera-Saldafia HA, Lambrou TP, Saunders G F (1982) Genome instability in a region of human DNA enriched in Alu repeat sequences. Nature 296:219225 Capon D J, Chen EY, Levinson AD, Seeburg PH, Goeddel DV (1983) Complete nucleotide sequences of the T24 h u m a n bladder carcinoma oncogene and its normal homologue. Nature 302:33-37 CattanachBM, RasberryC, BeecheyC (1990) Factors affecting mutation induction by x-rays in the spermatogonial stem cells of mice of strain 101/H. In: Banbury report 34: biology of mammalian germ cell mutagenesis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 209-220 Cedar H (1988) D N A methylation and gene activity. Cell 53: 3-4 Cera C, Pair G, Marciani Magno S, Palumbo M (1991) Interaction between second generation anthracyclines and D N A in nucleosomal structure. Nucleic Acids Res 19:2309-2314 Chandley AC, Kofman-Alfaro S (1971) "Unscheduled" DNA synthesis in h u m a n germ cells following U V irradiation. Exp Cell Res 69:45-48 Chandley AC, Mitchell AR (1988) Hypervariable minisatellite regions are sites for crossing-over at meiosis in man. Cytogenet Cell Genet 48:152-155 Chang LMS (t973) Low molecular weight deoxyribonucleic acid polymerase from calf thymus chromatin. J Biol Chem 248:6983-6992 Chary P, Mitchell CE, Kelly G (1991) Chemical carcinogeninduced damage to the c-neu and c-myc protooncogenes in rat lung epithelial cells: possible mechanisms for differential repair in transcriptionally active genes. Cancer Lett 58:57-63 C h e n R - H , M a h e r V M , MeCormickJJ (1990) Effect ofexcision repair by diploid h u m a n fibroblasts on the kinds and locations of mutations induced by (-+)-7fl,8a-dihydroxy-9a, 10a-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene in the coding region of the HPRT gene. Proe Natl Acad Sci USA 87:8680-8684 Clark DJ, Felsenfeld G (1991) Formation of nucleosomes on positively supercoiled DNA. EMBO J 10:387-395 Clark SP, Lewis CD, Felsenfeld G (1990) Properties of BGP1, a poly(dG)-binding protein from chicken erythrocytes. Nucleic Acids Res 18:5119-5126 Cleaver JE (1977) Nucleosome structure controls rates of excision repair in DNA of h u m a n cells. Nature 270:451-453 Cleaver JE (1978) DNA repair and its coupling to D N A replication in eukaryotic cells. Biochim Biophys Acta 516:489516 C l e r m o n t Y (1972) Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev 52:198-236 Cole KD, Kandala JC, Kistler WS (1986) Isolation of the gene

174 for the testis-specific H1 histone variant Hit*. J Biol Chem 261:7178-7183 Cooper DN, Krawczak M (1989) Cytosine methylation and the fate o f C p G dinucleotides in vertebrate genomes. Hum Genet 83:181-188 Cordeiro-Stone M, Topal MD, Kaufman D G (1982) D N A in proximity to the site of replication is preferentially alkylated in S phase 10T 1/2 cells treated with N-methyl-N-nitrosourea. Carcinogenesis 3:1119-1127 Corominas M, Mezquita C (1985) Poly(ADP-ribosyl)ation at successive stages of rooster spermatogenesis: levels of polymeric ADP-ribose in vivo and poly(ADP-ribose) polymerase activity and turnover of ADP-ribosyl residues in vitro. J Biol Chem 260:16269-16273 Cross CE, Halliwell B, Borish ET, Pryor WA, Ames BN, Saul RL, McCord JM, Harman D (1987) Oxygen radicals and h u m a n disease. Ann Int Med 107:526-545 CsordasA (1990) On the biological role ofhistone acetylation. Biochem J 265:23-38 C u t l e r R G (1975) Evolution o f h u m a n longevity and the genetic complex governing aging rats. Proc Natl Acad Sci USA 72: 4664-4668 Darzynkiewicz Z, Gledhill BL, Ringertz NR (1969) Changes in deoxyribonucleoprotein during spermiogenesis in the bull. 3Hactinomycin D binding capacity. Exp Cell Res 58:435-438 Davidson RL, Broeker P, Ashman CR (1988) DNA base sequence changes and sequence specificity of bromodeoxyuridine-induced mutations in mammalian ceils. Proc Natl Acad Sci USA 85:4406-4410 Davie JR, Murphy LC (1990) Level of ubiquitinated histone H2B in chromatin is coupled to ongoing transcription. Biochemistry 29:4752-4757 De Bernardin W, Koller T, Sogo JM (1986) Structure of an in vivo transcribing chromatin as studied in SV40 minichromosomes. J Mol Biol 191:469-482 de Cock JGR, Klink EC, Ferro W, Lohman PHM, Eeken JCJ (1991) Repair of UV-induced pyrimidine dimers in the individual genes Gart, Notch and white from Drosophila melanogaster cell lines. Nucleic Acids Res 19:3289-3294 Deutsch WA, Linn S (1979) DNA binding activity from cultured human fibroblasts that is specific for partially depurinated D N A and that inserts purines into apurinic sites. Proc Natl Acad Sci USA 76:141-144 Dianov G, Lindahl T (1991) Preferential recognition of I.T base-pairs in the initiation of excision-repair by hypoxanthine-DNA glycosylase. Nucleic Acids Res 19:3829-3833 Dickerson RE (1971) The structure of cytochrome c and the rates of molecular evolution. J Mol Evol 1:26-45 Dipple A, Pigott M, Moschel RC, Costantino N (1983) Evidence that binding of 7,12-dimethylbenz[a]anthracene to DNA in mouse embryo cell cultures results in extensive substitution of both adenine and guanine residues. Cancer Res 43:41324135 Dobzhansky T (1970) Genetics of the evolutionary process. Columbia University Press, New York Dolan ME, Oplinger M, Pegg AE (1988) Sequence specificity of guanine alkylation and repair. Carcinogenesis 9:2139-2143 Dorbic T, Wittig B (1987) Chromatin from transcribed genes contains HMG17 only downstream from the starting point of transcription. EMBO J 6:2393-2399 Drobetsky EA, Grosovsky AJ, Glickman BW (1987) The specificity of UV-induced mutations at an endogenous locus in mammalian cells. Proc Nail Acad Sci USA 84:9103-9107 Dunnick W, Stavnezer J (1990) Copy choice mechanism of immunoglobulin heavy-chain switch recombination. Mol Cell Biol 10:397-400 Durante M, Geri C, Bonatti S, Parenti R (1989) Non-random

alkylation of D N A sequences induced in vivo by chemical mutagens. Carcinogenesis 10:1357-1361 EcholsH (1981) SOS functions, cancer and inducible evolution. Cell 25:1-2 Echols H, Goodman MF (1991) Fidelity mechanisms in DNA replication. Ann Rev Biochem 60:477-511 Einck L, Bustin M (1983) Inhibition of transcription in somatic cells by microinjection of antibodies to chromosomal proteins. Proc Natl Acad Sci USA 80:6735-6739 Favor J (1990) Toward an understanding of the mechanisms of mutation induction by ethylnitrosourea in germ cells of the mouse. In: Banbury report 34: biology of mammalian germ cell mutagenesis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 221-235 Fawcett DW (1975) The mammalian spermatozoon. Dev Biol 44:394-436 Filipski J (1987) Correlation between molecular clock ticking, codon usage, fidelity of DNA repair, chromosome banding and chromatin compactness in germline cells. FEBS Lett 217: 184-186 Foster PL, Eisenstadt E, Miller JH (1983) Base substitution mutations induced by metabolically activated aflatoxin BI. Proc Natl Acad Sci USA 80:2695-2698 Frappier L, Price GB, Martin RG, Zannis-Hadjopoulos M (1989) Characterization of the binding specificity of two anticruciform D N A monoclonal antibodies. J Biol Chem 264: 334-341 Frederico LA, Kunkel TA, Shaw BR (1990) A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy. Biochemistry 29:2532-2537 GanesanA, SpivakG, HanawaltPC (1983) ExpressionofDNA repair genes in mammalian cells. In: Nagley P, Linnane AW, Peacock WJ, Pateman JA (eds) Manipulation and expression of genes in eukaryotes. Academic Press, New York, pp 45-54 Gatewood JM, Cook GR, Balhorn R, Bradbury EM, Schmid CW (1987) Sequence-specific packaging of D N A in h u m a n sperm chromatin. Science 236:962-964 Gatewood JM, Cook GR, Balhorn R, Schmid CW, Bradbury EM (1990) Isolation of four core histones from h u m a n sperm chromatin representing a minor subset of somatic histones. J Biol Chem 265:20662-20666 Generoso W M (1980) Repair in fertilized eggs of mice and its role in the production of chromosomal aberrations. Basic Life Sci 15:411-420 Generoso WM, Cain KT, Krishna M, HuffSW (1979) Genetic lesions induced by chemicals in spermatozoa and spermatids of mice are repaired in the egg. Proc Natl Acad Sci USA 76: 435-437 Giaever GN, Wang JC (1988) Supercoiling ofintracellular DNA can occur in eukaryotic cells. Cell 55:849-856 Gilmour RS, Spandidos DA, Vass JK, Gow JW, Paul J (1984) A negative regulatory sequence near the mouse/%maj globin gene associated with a region of potential Z-DNA. EMBO J 3:1263-1272 Giloni L, Take shita M, Johnson F, Iden C, Grollman AP (1981) Bleomycin-induced strand-scission of DNA. Mechanism of deoxyribose cleavage. J Biol Chem 256:8608-8615 Glickman BW, Schaaper RM, Haseltine WA, Dunn RL, Brash DE (1986) The C-C (6-4) U V photoproduct is mutagenic in Escherichia coli. Proc Natl Acad Sci USA 83:6945-6949 Gojobori T, Li W-H, Graur D (1982) Patterns of nucleotide substitution in pseudogenes and functional genes. J Mol Evol 18:360--369 G o o d m a n M (1981) Globin evolution was apparently very rapid in early vertebrates: a reasonable case against the rateconstancy hypothesis. J Mol Evol 17:114-120 Govan HL III, Valles-Ayoub Y, Braun J (1990) Fine-mapping

175 of DNA damage and repair in specific genomic segments. Nucleic Acids Res 18:3823-3830 Greer WL, Kaplan J G (1986) Early nuclear events in lymphocyte proliferation: the role of DNA strand break repair and ADP ribosylation. Exp Cell Res 166:399-415 Grimes S, Weisz-Carrington P, Daum H I I I , Smith J, Green L, Wright K, Stein G, Stein J (1987) A rat histone H4 gene closely associated with the testis-specific H 1t gene. Exp Cell Res 173:534-545 Grosovsky A J, de Boer JG, de Jong PJ, Dobetsky EA, Glickman BW (1988) Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian celts. Proc Natl Acad Sci USA 85:185-188 Grossman L, Yeung AT (1990) The U v r ABC endonuclease of Escherichia coli. Photochem Photobiol 51:749-755 Guli CL, Smyth DR (1988) UV-induced DNA repair is not detectable in predictyate oocytes of the mouse. Mutat Res 208:115-119 Guli CL, Smyth DR (1989) Lack of effect ofmaterual age on UV-induced D N A repair in mouse oocytes. Mutat Res 210: 323-328 Gusse M, Sautiere P, Belaiche D, Martinage A, Roux C, Dadoune J-P, Chevaillier P (1986) Purification and characterization of nuclear basic proteins of h u m a n sperm. Biochim Biophys Acta 884:124-134 H a h n BH, Shaw GM, Taylor ME, Redfield RR, Markham PD, Salahuddin SZ, Wong-Staal F, Gallo RC, Parks ES, Parks WP (1986) Genetic variation in HTLV-III/LAV over time in patients with AIDS or at risk for AIDS. Science 232:15481553 Hall KY, Hart RW, Benirschke AK, Walford RL (1984) Correlation between ultraviolet-induced DNA repair in primate lymphocytes and fibroblasts and species maximum achievable life span. Mech Ageing Dev 24:163-173 Hanawalt PC (1989) Preferential repair of damage in actively transcribed D N A sequences in vitro. Genome 31:605-611 Hanawalt PC (1991) Heterogeneity of DNA repair at the gene level. Mutat Res 247:203-211 Hart RW, Modak SP (1980) Aging and changes in genetic information. Adv Exp Med Biol 129:123-137 Hart RW, Setlow RB (1974) Correlation between deoxyribonucleic acid excision-repair and life-span in a number of mammalian species. Proc Natl Acad Sci USA 71:2169-2173 Hart RW, D'Ambrioso SM, Ng KJ, Modak SP (1979) Longevity, stability, and DNA repair. Mech Ageing Dev 9:203-223 Hartley JA, Gibson NW, Kohn KW, Mattes WB (1986) D N A sequence selectivity of guanine-N7 alkylation by three antitumor chloroethylating agents. Cancer Res 46:1943-1947 Harwood J, Tachibana A, Meuth M (1991) Multiple dispersed spontaneous mutations: a novel pathway of mutation in a malignant h u m a n cell line. Mol Cell Biol 11:3163-3170 H e c h t N B (1989) Molecular biology ofstructural chromosomal proteins of the mammalian testis. In: Adolph KW (ed) Molecular biology of chromosome function. Springer-Verlag, New York, pp 396-420 Helland DE, Male R, Haukanes BI, Olsen L, Haugan I, Kleppe K (1987) Properties and mechanism of action of eukaryotic 3-methyladenine-DNA glycosylases. J Cell Sci Suppl 6:139146 Herskovits TT, Brahms J (1976) Structural investigations on DNA.protamine complexes. Biopolymers 15:687-706 Hinds KR, Litman G W (1986) Major reorganization of immunoglobulin V H segmental elements during vertebrate evolution. Nature 320:546-549 Ho L, Bohr VA, Hanawalt PC (1989) Demethylation enhances removal of pyrimidine dimers from the overall genome and from specific DNA sequences in Chinese hamster ovary cells. Mol Cell Biol 9:1594-1603

Hoeijmakers JHJ, Bootsma D (1990) Molecular genetics of eukaryotic D N A excision repair. Cancer Cells 2:311-320 Hogan ME, Rooney TF, Austin R H (1987) Evidence for kinks in D N A folding in the nucleosome. Nature 328:554-557 Holmquist G P (1987) Role of replication time in the control of tissue-specific gene expression. A m J Hum Genet 40:151173 Holmquist G P (1989) Evolution of chromosome bands: molecular ecology of noncoding DNA. J Mol Evol 28:469--486 Homberger HP (1989) Bent DNA is a structural feature of scaffold-attached regions in Drosophila melanogaster interphase nuclei. Chromosoma 98:99-104 Horimoto K, Suzuki H, Otsuka J (1990) Discrimination between adaptive and neutral amino aeid substitutions in vertebrate hemoglobins. J Mol Evol 31:302-324 Hsu IC, Metealf RA, Sun T, Welsh JA, Wang NJ, Harris CC (199t) Mutational hotspot in the p53 gene in h u m a n hepatocellular carcinomas. Nature 350:427--428 Hu H, Ray PN, Worton R G (1991) Mechanisms of tandem duplication in the Duchenne muscular dystrophy gene include both homologous and nonhomologous intrachromosomal recombination. EMBO J 10:2471-2477 Hurwitz J, Dean FB, Kwong AD, Lee S-H (1990) The in vitro replication of D N A containing the SV40 origin. J Biol Chem 265:18043-18046 Hutchinson R (1985) Chemical changes induced in DNA by ionizing radiation. Prog Nucleic Acids Res Mol Biol 32:115154 Jeffreys AJ, Wilson V, Thein SL (1985) Hypervariable "minisatellite" regions in h u m a n DNA. Nature 314:67-73 Jensen KA, Smerdon MJ (1990) DNA repair within nucleosome cores of UV-irradiated h u m a n cells. Biochemistry 29: 4773--4782 J o h n s t o n B H (1988) TheSl-sensitiveformofd(C-T)n.d(A-G)n: chemical evidence for a three-stranded structure in plasmids. Science 241:1800-1804 Johnston BH, Rich A (1985) Chemical probes of D N A conformation: detection of Z-DNA at nucleotide resolution. Cell 42:713-724 Johnston BH, Quigley GJ, Ellison MJ, Rich A (1991) The Z-Z junction: the boundary between two out-of-phase Z-DNA regions. Biochemistry 30:5257-5263 Jones CW, Kafatos FC (1982) Accepted mutations in a gene family: evolutionary diversification of duplicated DNA. J Mol Evol 19:87-103 Jurka J, Smith T (1988) A fundamental division in the Alu family of repeated sequences. Proc Natl Acad Sci USA 85: 4775--4778 Kelley MR, Venugopal S, Harless J, Deutsch WA (1989) Antibody to a h u m a n D N A repair protein allows for cloning of a Drosophila cDNA that encodes an apurinic endonuclease. Mol Cell Biol 9:965-973 Kierszenbaum AL, Tres LL (1975) Structural and transcriptional features of the mouse spermatid genome. J Cell Biol 65:258-270 Kim Y-J, Hwang I, Tres LL, Kierszenbaum AL, Chae C-B (1987) Molecular cloning and differential expression of somatic and testis-specific H2B histone genes during rat spermatogenesis. Dev Biol 124:23-34 Kimura MS (1969) The rate of molecular evolution considered from the standpoint of population genetics. Proc Natl Acad Sci USA 63:1181-1188 Kimura MS (1983) The neutral theory of molecular evolution. Cambridge University Press, New York Kimura MS (1989) The neutral theory of molecular evolution and the world view of the neutralists. Genome 31:24-31 Kimura MS, Ohta T (1973) Mutation and evolution at the molecular level. Genetics Suppl 73:19-35

176 Klein F, Karwan A, Wintersberger U (1989) After a single treatment with EMS the number of non-colony-forming cells increases for many generations in yeast populations. Mutat Res 210:157-164 Klein F, Karwan A, Wintersberger U (1990) Pedigree analyses of yeast cells recovering from DNA damage allow assignment of lethal events to individual post-treatment generations. Genetics 124:57-65 Klein J (1978) H-2 mutations: their genetics and effect on immune functions. J Adv Immunol 26:55-147 Klein J (1979) The major histoeompatibility complex of the mouse. Science 203:516-521 Koehler DR, Awadallah SS, Glickman BW (1991) Sites of preferential induction of cyclobutane pyrimidine dimers in the nontranscribed strand of lac I correspond with sites of UVinduced mutation in Escherichia coli. J Biol Chem 266:1176611773 Kohne DE (1970) Evolution of higher-organism DNA. Quart Rev Biophys 33:327-375 Kohwi Y (1989) Non-B-DNA structure: preferential target for the chemical carcinogen glycidaldehyde. Carcinogenesis 10: 2035-2042 Kohwi-Shigematsu T, Gelinas R, Weintraub H (1983) Detection of an altered D N A conformation at specific sites in chromatin and supercoiled DNA. Proc Natl Acad Sci USA 80: 4389-4393 Kunisawa T, Horimoto K, Otsuka J (1987) Accumulation pattern of amino acid substitutions in protein evolution. J Mol Evol 24:357-365 Kunkel GR, Martinson H G (1981) Nucleosomes will not form on double-stranded RNA or over poly(dA), poly(dT) tracts in recombinant DNA. Nucleic Acids Res 9:6869-6888 LeDoux SP, Patton NJ, Nelson JW, Bohr VA, Wilson GL (1990) Preferential D N A repair of alkali-labile sites within the active insulin gene. J Biol Chem 265:14875-14880 Lee M-S, Garrard WT (1991) Transcription-induced nucleosome 'splitting': an underlying structure for DNase I senstive ehromatin. EMBO J 10:607-615 Lehrman MA, Russell DW, Goldstein JL, Brown MS (1987) Alu-Alu recombination deletes splice acceptor sites and produces secreted low density lipoprotein receptor in a subject with familial hypercholesterolemia. J Biol Chem 262:33543361 Lemaire MA, Schwartz A, Rahmouni AR, Leng M (1991) Interstrand cross-links are preferentially formed at the d(GC) sites in the reaction between eis-diaminedichloroplatinum(II) and DNA. Proe Natl Aead Sei USA 88:1982-1985 Li W-H, Graur D (1991) Fundamentals of molecular evolution. Sinauer, Sunderland MA Li W-H, Wu C-I, Luo C-C (1984) Nonrandomness of point mutation as reflected in nueleotide substitutions in pseudogenes and its evolutionary implications. J Mol Evol 21:58-71 Lindahl T (1982) DNA repair enzymes. Annu Rev Bioehem 51:61-87 Lindahl T (1990) Repair of intrinsic DNA lesions. Murat Res 238:305-311 Lindahl T, Ljungquist S, Siegert W, Nyberg B, Sperens B (1977) D N A N-glyeosidases. Properties of uracil-DNA glycosidase from Escheriehia coli. J Biol Chem 252:3286-3294 Liu S-C, Parsons S, Hanawalt PC (1983) D N A repair in cultured keratinoeytes. J Invest Dermatol 81:179s--183s Livneh Z, Elad D, Sperling J (1979) Enzymatic insertion of purine bases into depurinated DNA in vitro. Proc Natl Aead Sci USA 76:1089-1093 Loeb LA, Kunkel TA (1982) Fidelity of DNA synthesis. Annu Rev Biochem 51:429-457 Loechler EL, Green CL, Essigmann JM (1984) In vivo mutagenesis by Or-methylguanine built into a unique site in a viral genome. Proc Nat1 Acad Sei USA 81:6271-6275

Losa R, Omari S, Thoma F (1990) Poly(dA).poly(dT) rich sequences are not sufficient to exclude nueleosome formation in a constitutive yeast promoter. Nucleic Acids Res 18:34953502 LovelessA (1969) PossiblerelevanceofO6-alkylationofdeoxy guanosine to the mutagenicity and careinogenicity of nitrosamines and nitrosamides. Nature 223:206-207 Ludlum DB (1970) Alkylated polycytidylic acid templates for RNA polymerase. Biochim Biophys Acta 213:142-148 LuriaSE, DelbriickM (1943) Mutations ofbacteria from virus sensitivity to virus resistance. Genetics 28:491-511 Lyamichev V (1991) Unusual conformation of (dA)n.(dT),tracts as revealed by cyelobutane thymine-thymine dimer formation. Nucleic Acids Res 16:4491-4496 Martinez-Cruzado JC (1990) Evolution of the autosomal chorion cluster in Drosophila. IV. The Hawaiian Drosophila: rapid protein evolution and constancy in the rate of D N A divergence. J Mol Evol 31:402-423 MatsudaY, T o b a r i I (1989) Repair capacity offertilized mouse eggs for x-ray damage induced in sperm and mature oocytes. Mutat Res 210:35-47 Matsuda Y, Seki N, Utsugi-Takeuchi T, Tobari I (1989a) Changes in x-ray sensitivity of mouse eggs from fertilization to the early pronuclear stage, and their repair capacity. Int J Radiat Biol 55:233-256 Matsuda Y, Maemori M, Tobari I (1989b) Relationship between cell cycle stage in the fertilized egg of mice and repair capacity for x-ray-induced damage in the sperm. Int J Radiat Biol 56:301-314 MatsudaY, SekiN, Utsugi-TakeuchiT, TobariI (1989c) X-rayand mitomyein C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the x-ray and MMC damage. Murat Res 211:65-75 Matsunami N, Hamaguchi Y, Yamamoto Y, Kuze K, Kangawa K, Matsuo H, Kawaichi M, Honjo T (1989) A protein binding to the J, recombination sequence of immunoglobulin genes contains a sequence related to the integrase motif. Nature 342:934-937 Mattes WB, Hartley JA, Kohn KW (1986) DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards. Nucleic Acids Res 14:2971-2987 Mattes WB, Hartley JA, Kohn KW, Matheson DW (1988) GCrich regions in genomes as targets for DNA alkylation. Carcinogenesis 9:2065-2072 Mavrothalassitis GJ, Watson DK, Papas TS (1990) Molecular and functional characterization of the promoter of ETS2, the h u m a n c-ets-2 gene. Proe Natl Acad Sci USA 87:1047-1051 Maxam, Gilbert (1977) A new method for sequencing DNA. Proe Natl Acad Sci USA 74:560-564 McGregor WG, Chen R-H, Lukash L, Maher VM, McCormick JJ (1991) Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient h u m a n fibroblasts but not in repair-deficient cells. Mol Cell Biol 11:1927-1934 Meistrich ML, Bucci LR, Trostle-Weige PK, Brock WA (1985) Histone variants in rat spermatogonia and primary spermatocytes. Dev Biol 112:230-240 Mellon I, Hanawalt PC (1989) Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand. Nature 342:95-98 Mellon I, Bohr VA, Smith CA, Hanawalt PC (1986) Preferential DNA repair of an active gene in h u m a n cells. Proc Natl Acad Sci USA 83:8878-8882 Mellon I, Spivak G, Hanawalt PC (1987) Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian D H F R gene. Cell 51:241-249 Merchant JL, Demediuk B, Brand SJ (1991) A GC-rich element confers epidermal growth factor responsiveness to transcription from the gastrin promoter. Mol Cell Biol 11:2686-2696

177 Mirzabekov AD, San'ko DF, Kolchinsky AM, Melnikova AF (1977) Protein arrangement in the DNA grooves in chromatin and nucleoprotamine in vitro and in vivo revealed by methylation. Eur J Biochem 75:379-389 Mirzabekov AD, Shick VV, Belyavsky AV, Bavykin SG (1978) Primary organization of nucleosome core particle of chromatin: sequence of histone arrangement along DNA. Proc Natl Acad Sci USA 75:4184--4188 Mitchell DL, Nairn RS (1989) The biology of the (6-4) photoproduct. Photochem Photobiol 49:805-819 Mitchell DL, Haipek CA, Clarkson J M (1985) (64)Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane pyrimidine dimers. Mutat Res 143:109-112 Mitchell DL, Nguyen TD, Cleaver JE (1990) Nonrandom induction of pyrimidine-pyrimidone (6-4) photoproducts in ultraviolet-irradiated human chromatin. J Biol Chem 265: 5353-5356 Mitchell N, Strhrer G (1986) Mutagenesis originating in sitespecific DNA damage. J Mol Biol 191:177-180 Modrich P (1987) DNA mismatch correction. Annu Rev Biochem 56:435--466 Montesano R (l 981) Alkylation of DNA and tissue specificity in nitrosamine carcinogenesis. J Supramol Struct Cell Biochem 17:259-273 Moynet D, MacLean SJ, Ng KH, Anctil D, Gibson DM (1985) Polymorphism of K variable region (VK-1) genes in inbred mice: relationship to the Igr-Ef2 serum light chain marker. J Immunol 134:3455-3460 Muench KF, Misra RP, Humayun MZ (1983) Sequence specificity in aflatoxin BI-DNA interactions. Proc Natl Acad Sci USA 80:6-10 Mullenders LHF, van Kesteren AC, Bussmann CJM, van ZeelandAA, NatarajanAT (1984) Preferential repair ofnuclear matrix associated DNA in xeroderma pigmentosum complementation group C. Mutat Res 141:75-82 Mullenders LHF, van Kesteren AC, van Leeuwen AC, van Zeeland AA, Natarajan AT (1988) Nuclear matrix associated DNA is preferentially repaired in normal human fibroblasts exposed to a low dose of ultraviolet light but not in Cockayne's syndrome fibroblasts. Nucleic Acids Res 16:10607-10623 Nacheva GA, Guschin DY, Preobrazhenskaya OV, Karpov VL, EbralidseKK, MirzabekovAD (1989) Change in the pattern of histone binding to DNA upon transcriptional activation. Cell 58:27-36 Nejedly K, Kwinkowski M, Galazka G, Klysik J, Palecek E (1985) Rec°gniti°n ° f the structural dist°rsi°ns at the junctions between B and Z segments in negatively supercoiled DNA by osmium tetroxide. J Biomol Struct Dyn 3:467-478 Nikaido T, Nakai S, Honjo T (1981) Switch region o f i m m u noglobulin C~ gene is composed of simple tandem repetitive sequences. Nature 292:845-848 Oesch F, Aulmann W, Platt KL, Doerjer G (1987) Individual differences in DNA repair capacities in man. Arch Toxicol Suppl 10:172-179 Ogura T, Takenouchi N, Yamaguchi M, Matsukage A, Sugimura T, Esumi H (1990) Striking similarity of the distribution patterns of the poly(ADP-ribose) polymerase and DNA polymerase/3 among various mouse organs. Biochem Biophys Res Commun 172:377-384 Ohta T (1991) Multigene families and the evolution of complexity. J Mol Evol 33:34-41 Okumoto DS, Bohr VA (1987) DNA repair in the metallothionein gene increases with transcriptional activation. Nucleic Acids Res 15:10021-10030 Panayotatos N, Fontaine A (1987) A native cruciform DNA structure probed in bacteria by recombinant T7 endonuclease. J Biol Chem 262:11364-11368 Pedersen RA, Brandriff B (1980) Radiation- and drug-induced

DNA repair in mammalian oocytes and embryos. Basic Life Sci 15:389-410 Pegg AE, Wiest L, Foote RS, Mitra S, Perry W (1983) Purification and properties of O6-methylguanine-DNA transmethylase from rat liver. J Biol Chem 258:2327-2333 Pegg AE, Scicchitano D, Dolan ME (1984) Comparison of the rates of repair of O6-alkylguanines in DNA by rat liver and bacterial O6-alkylguanine-DNA alkyltransferase. Cancer Res 44:3806-3811 Peters J, Jones J, Ball ST, Clegg JB (1990) Analysis ofelectrophoretically detected mutations induced in mouse germ cells by ethylnitrosourea. In: Banbury report 34: biology of mammalian germ cell mutagenesis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 247-257 Pfeifer GP, Drouin R, Riggs AD, Holmquist GP (1991) In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6-4) pyrimidone photoproducts by ligationmediated polymerase chain reaction. Proc Natl Acad Sci USA 88:1374-1378 Planche-Martel G, Likhachev A, Wild CP, Montesano R (1985) Modulation of repair of O6-methylguanine in parenchymal and nonparenchymal liver cells of rats treated with dimethylnitrosamine. Cancer Res 45:4768-4773 Powell JR (1971) Genetic polymorphism in varied environments. Science 174:1035-1036 Preston BD, Poiesz BJ, Loeb LA (1988) Fidelity of HIV-1 reverse transcriptase. Science 242:1168-1171 Protic-Sabljic M, Tuteja N, Munson PJ, Hauser J, Kraemer KH, Dixon K (1986) UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells. Mol Cell Biol 6: 3349-3356 Prunell A (1982) Nucleosome reconstitution on plasmid-inserted poly(dA), poly(dT). EMBO J 1:173-179 Pu WT, Kahn R, Munn M, Rupp WD (1989) Uvr ABC incision ofN-methylmitomycin A - D N A monoadducts and cross-links. J Biol Chem 264:20697-20704 Raha M, Hutchinson F (1991) Deletions induced by gamma rays in the genome of Escherichia coli. J Mol Biol 220:193198 Ramanathan B, Smerdon MJ (1989) Enhanced DNA repair synthesis in hyperacetylated nucleosomes. J Biol Chem 264: 11026-11034 Ramanathan R, Rajalakshmi S, Sarma DSR, Farber E (1976) Nonrandom nature of in vivo methylation by dimethylnitrosamine and the subsequent removal of methylated products from rat liver chromatin DNA. Cancer Res 36:2073-2079 Rao BS, Zannis-Hadjopoulos M, Price GB, Reitman M, Martin RG (1990) Sequence similarities among monkey ori-enriched (ors) fragments. Gene 87:233-242 Razin SV, Vassetzky YS, Hancock R (1991) Nuclear matrix attachment regions and topoisomerase II binding and reaction sites in the vicinity of a chicken DNA replication origin. Biochem Biophys Res Commun 177:265-270 ReinerB, ZamenhofS (1957) Studies on the chemically reactive groups of deoxyribonucleic acids. J Biol Chem 228:475-476 Reneker JS, Brothelton TW (1991) Discrete regions of the avian/3-globin gene cluster have tissue-specific hypersensitivity to cleavage by sonication in nuclei. Nucleic Acids Res 19: 4739-4745 Rhodes C, Savagner P, Line S, Sasaki M, Chirigos M, Doege K, Yamada Y (199 I) Characterization of the promoter for the rat and human link protein gene. Nucleic Acids Res 19:19331939 Richard-FoyH, HagerGL (1987) Sequence-specificpositioning of nucleosomes over the steroid-inducible MMTV promoter. EMBO J 6:2321-2328 Richardson KK, Richardson FC, Crosby RM, Swenberg JA, Skopek TR (1987) DNA base changes and alkylation following in vivo exposure of Escherichia coli to N-methyl-N-nitroso-

178 urea or N-ethyl-N-nitrosourea. Proc Natl Acad Sci USA 84: 334-348 Riley MA (1989) Nucleotide sequence of the Xdh region in Drosophila pseudoobscura and an analysis of the evolution of synonymous codons. Mot Biol Evol 6:33-52 Roberts JD, Thomas DC, Kunkel TA (1991) Exonucleolytie proofreading of leading and lagging strand DNA replication errors. Proc Natl Acad Sci USA 88:3465-3469 Robson CN, Milne AM, Pappin DJC, Hickson ID (1991) Isolation o f c D N A clones encoding an enzyme from bovine cells that repairs oxidative DNA damage. In vitro homology with bacterial repair enzymes. Nucleic Acids Res 19:1087-1092 Rolfe M, Spanos A, Banks G (1986) Induction of yeast Ty element transcription by ultraviolet light. Nature 319:339340 Roth SY, Dean A, Simpson RT (1990) Yeast a2 repressor positions nucleosomes in TRP1/ARS1 chromatin. Mol Cell Biol 10:2247-2260 RussellWL (1956) Comparison ofx-ray induced mutation rates in Drosophila and mice. A m Nat 90:69 RussevG, BoulikasT (1992) Repair of transcriptionally active and inactive genes during S and G2 phases of the cell cycle. Eur J Biochem 204:267-272 Russev G, Vassilev L (1982) Isolation o f a DNA fraction from Ehrlich ascites tumour cells containing the putative origin of replication. J Mol Biol 161:77-87 Ryan AJ, Billett MA, O'Connor PJ (1986) Selective repair of methylated purines in regions of chromatin DNA. Carcinogenesis 7:1497-1503 SakanoH, HiippiK, HeinrichG, TonegawaS (1979) Sequences at the somatic recombination sites of immunoglobulin lightchain genes. Nature 280:288-294 Sakumi K, Sekiguehi M (1990) Structures and functions of DNA glycosylases. Mutat Res 236:161-172 SaragostiS, MoyneG, Y a n i v M (1980) Absenceofnucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA. Cell 20:65-73 Schaaper RM, Dunn RL, Glickman BW (1987) Mechanisms of ultraviolet-induced mutation. Mutational spectra in the Escheriehia coli lac I gene for a wild-type and an excisionrepair-deficient strain. J Mol Biol 198:187-202 Schatz DG, Oettinger MA, Baltimore D (1989) The V(D)J recombination activating gene, RAG- 1. Cell 59:1035-1048 Schlissel MS, Baltimore D (1989) Activation of immtmoglobulin kappa gene rearrangement correlates with induction of germline kappa gene transcription. Cell 58:1001 - 1007 Scicchitano DA, Hanawalt PC (1989) Repair of N-methylpufines in specific DNA sequences in Chinese hamster ovary cells: absence of strand specificity in the dihydrofolate reductase gene. Proc Natl Acad Sci USA 86:3050-3054 Sega G A (1974) Unscheduled DNA synthesis in the germ cells of male mice exposed in vivo to the chemical mutagen ethyl methanesulfonate. Proc Natl Acad Sci USA 71:4955-4959 Sega G A (1990) Molecular targets, DNA breakage, and DNA repair: their roles in mutation induction in mammalian germ cells. In: Banbury report 34: biology of mammalian germ cell mutagenesis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 79-91 Seidman JG, Max EE, Leder P (1979) A K-immunoglobulin gene is formed by site-specific recombination without further somatic mutation. Nature 280:370-375 Seidman M, Slor H, Bustin M (1983) The binding of a carcinogen to the nucleosomal and non-nucleosomal regions of the simian virus 40 chromosome in vivo. J Biol Chem 258:52155220 Sekiya T, Kobayashi M, Seki T, Koike K (1980) Nucleotide

sequence of a cloned fragment of rat mitochondrial DNA containing the replication origin. Gene 11:53-62 SelbyCP, SancarA (1990) Structure and function o f t h e (A)BC excinuclease of Escheriehia coli. Mutat Res 236:203-211 Sen R, Baltimore D (1986) Multiple nuclear factors interact with the immunoglobin enhancer sequences. Cell 46:705-716 Setlow JK, Setlow RB (1963) Nature of the photoreaetivable ultraviolet lesion in DNA. Nature 197:560 Seyedin SM, Cole RD, Kistler WS (1981) H1 histones from mammalian testes. Exp Cell Res 136:399-405 ShiresA, CarpenterMP, ChalkleyR (1975) New histones found in mature mammalian testes. Proc Natl Acad Sci USA 72: 2714-2718 Simpson RT (1986) Nucleosome positioning in vivo and in vitro. BioEssays 4:172-176 Simpson RT, Kfinzler P (1979) Chromatin and core particles formed from the inner histones and synthetic polydeoxyribonucleotides of defined sequence. Nucleic Acids Res 6:13871415 Singer B (1976) All oxygens in nucleic acids react with carcinogenic ethylating agents. Nature 264:333-339 Smerdon MJ, T h o m a F (t990) Site-specific D N A repair at the nucleosome level in a yeast minichromosome. Cell 6 t :675684 Smith GR, Kunes SM, Schultz DW, Taylor A, Triman KL (1981) Structure of Chi hotspots of generalized recombination. Cell 24:429-436 Snow ET, Foote RS, Mitra S (1984) Base-pairing properties of O6-methylguanine in template D N A during in vitro DNA replication. J Biol Chem 259:8095-8100 Sogo JM, Stahl H, Koller T, Knippers R (1986) Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures. J Mol Biol 189:189-204 Solomon MJ, Larsen PL, Varshavsky A (1988) Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell 53:937-947 Souliotis VL, Giannopoulos A, Koufakis I, Kaila S, Dimopoulos C, Kyrtopoulos SA (1989) Development and validation of a new assay for O6-alkylguanine-DNA-alkyltransferase based on the use of an oligonucleotide substrate, and its application to the measurement of DNA repair activity in extracts of biopsy samples of h u m a n urinary bladder mucosa. Carcinogenesis I0:1203-1208 Steinemann M (1981) Chromosomal replication in Drosophila virilis. III Organization of active origins in the high polytene salivary gland cells. Chromosoma 82:289-307 Steinmetz M, Stephan D, Lindahl KF (1986) Gene organization and recombinational hotspots in the murine major histocompatibility complex. Cell 44:895-904 Stewart C-B, Schilling JW, Wilson AC (1987) Adaptive evolution in the stomach lysozymes of foregut fermenters. Nature 330:401-404 Stillman B (1989) Initiation of eukaryotic D N A replication in vitro. Annu Rev Cell Biol 5:197-245 Straka C, H6rz W (1991) A functional role for nucleosomes in the repression of a yeast promoter. EMBO J 10:361-368 Strand DJ, McDonald J F (1985) Copia is transcriptionally responsive to environmental stress. Nucleic Acids Res 13:44014410 Suau P, Subirana JA (1977) X-ray diffraction studies of nucleoprotamine structure. J Mol Biol 117:909-926 Sugino A, Drake JW (1984) Modulation of mutation rates in bacteriophage T4 by a base-pair change a dozen nucleotides removed. J Mol Biol 176:239-249 Tanaka K, Miura N, Satokata I, Miyamoto I, Yoshida MC, Satoh

179 Y, Kondo S, Yasui A, Okayama H, Okada Y (1990) Analysis of a h u m a n D N A excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain. Nature 343:73-76 TanakaT, N e i M (1989) Positive Darwinian selection observed at the variable region genes of immunoglobulins. Mol Biol Evol 6:447-459 Tanphaichitr N, Sobhon P, Taluppeth N, Chalermisarachai P (1978) Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man. Exp Cell Res 117:347-356 Terleth C, van Sluis CA, van de Putte P (1989) Differential repair of U V damage in Saccharomyces cerevisiae. Nucleic Acids Res 17:4433-4439 Terleth C, Waters R, Brouwer J, van de Putte P (1990) Differential repair of U V damage in Saccharomyces cerevisiae is cell cycle dependent. EMBO J 9:2899-2904 ThackerJ (1985) The molecular nature ofmutations in cultured mammalian cells: a review. Murat Res 150:431-442 Thackeray JR, Kyriacou CP (1990) Molecular evolution in the Drosophila yakuba period locus. J Mol Evol 31:389-401 T h o m a F , S i m p s o n R T (1985) Local protein-DNA interactions may determine nucleosome positions on yeast plasmids. Nature 315:250-252 Thomas DC, Roberts JD, Kunkel TA (1991) Heteroduplex repair in extracts of h u m a n HeLa cells. J Biol Chem 266: 3744-3751 Ticher A, Graur D (1989) Nucleic acid composition, codon usage, and the rate of synonymous substitution in proteincoding genes. J Mol Evol 28:286-298 Topal MD (1988) DNA repair, oncogenes and carcinogenesis. Carcinogenesis 9:691-696 Topal MD, Eadie JS, Conrad M (1986) O6-methylguanine mutation and repair is nonuniform. Selection for DNA most interactive with O6methylguanine. J Biol Chem 261:98799885 Travers AA (1987) D N A bending and nucleosome positioning. Trends Biochem Sci 12:108-112 Trifonov EN (1986) Curved DNA. CRC Crit Rev Biochem 19: 89-106 Trifonov EN (1989) Viewpoint: the multiple codes of nucleotide sequences. Bull Math Biol 51:417-432 Trifonov EN, Mengeritsky G (1987) Bent DNA in chromatin versus free curved DNA. In: Olson WK, Sarma MH, Sarma RH, Sundaralingam M (eds) Structure and expression, vol 3: D N A bending and curvature. Adenine Press, Guilderland, NY, pp 159-167 Tsao Y-P, Wu H-Y, Liu LF (1989) Transcription-driven supercoiling of DNA: direct biochemical evidence from in vitro studies. Cell 56:111-118 U p h o l t W B , DawidIB (1977) M a p p i n g o f m i t o c h o n d r i a l D N A of individual sheep and goats: rapid evolution in the D loop region. Cell 11:571-583 van Houten B (1990) Nucleotide excision repair in Escherichia coli. Microbiol Rev 54:18-51 Varshavsky A J, Sundin O, Bohn M (1979) A stretch o f " l a t e " SV40 viral D N A about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes. Cell 16:453-466 Venema J, Mullenders LHF, Natarajan AT, van Zeeland AA, Mayne LV (1990a) The genetic defect in Cockayne syndrome is associated with a defect in repair of UV-induced DNA damage in transcriptionally active DNA. Proc Natl Acad Sci USA 87:4707-4711 Venema J, van Hoffen A, Natarajan AT, van Zeeland AA, Mullenders LHF (1990b) The residual repair capacity of xeroderma pigmentosum complementafion group C fibroblasts is highly specific for transcriptionally active DNA. Nucleic Acids Res 18:443-448

Venema J, van Hoffen A, Karcagi V, Natarajan AT, van Zeeland AA, Mullenders LHF (1991) Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes. Mol Cell Biol 11:4128-4134 Vrieling H, Van Rooijen ML, Groen NA, Zdzienicka MZ, Simons JWIM, Lohman PHM, van Zeeland AA (1989) D N A strand specificity for UV-induced mutations in mammalian cells. Mol Cell Biol 9:1277-1283 Vrieling H, Venema J, van Rooyen M-L, van Hoffen A, Menichini P, Zdzienicka MZ, Simons JWIM, Mullenders LHF, van Zeeland AA (1991) Strand specificity for UV-induced DNA repair and mutations in the Chinese hamster H P R T gene. Nucleic Acids Res 19:2411-2415 Wahls WP, Wallace LJ, Moore PD (1990) Hypervariable minisatellite D N A is a hotspot for homologous recombination in h u m a n cells. Cell 60:95-103 Wahls WP, Swenson G, Moore PD (1991) Two hypervariable minisatellite DNA binding proteins. Nucleic Acids Res 19: 3269-3274 Walker MS, Becker FF, Rodriguez LV (1979) In vivo binding of N-2-acetylaminofluorene and its N-hydroxy derivative to the D N A of fractionated rat liver chromatin. Chem Biol Interactions 27:177-190 Watson JD, Crick FI-IC (1953) The structure of DNA. Cold Spring Harbor Symp Quant Biol 18:123-131 Weber CA, Salazar EP, Stewart SA, Thompson LH (1990) ERCC-2--cDNA cloning and molecular characterization of a h u m a n nucleotide excision repair gene with high homology to yeast Rad3. EMBO J 9:1437-1447 Weeda G, van Ham RCA, Vermeulen W, Bootsma D, van der Eb AJ, Hoeijmakers JHJ (1990) A presumed DNA helicase encoded by the excision repair gene ERCC-3 is involved in the h u m a n repair disorders xeroderma pigmentosum and Cockayne's syndrome. Cell 62:777-791 Weller SK, Spadaro A, Schaffer JE, Murray AW, Maxam AM, Schaffer PA (1985) Cloning, sequencing, and functional analysis of oriL, a herpes simplex virus type 1 origin of DNA synthesis. Mol Cell Biol 5:930-942 WiebauerK, JiricnyJ (1990) Mismatch-specificthymineDNA glycosylase and DNA polymerase/3 mediate the correction of G. T mispairs in nuclear extracts from h u m a n cells. Proc Natl Acad Sci USA 87:5842-5845 Wilkins RJ, Hart RW (1973) Preferential DNA repair in h u m a n cells. Nature 247:35-36 WilsonAC, CarsonSS, WhiteSJ (1977) Biochemicalevolution. Annu Rev Biochem 46:573-639 WintersbergerU (1984) The selective advantage ofcancer cells: a consequence of genome mobilization in the course of the induction of DNA repair processes? (Model studies on yeast). Adv Enzyme Regul 22:311-323 WintersbergerU (1991) On the origins ofgenetic variants. FEBS Lett 2:160-164 Wiseman RW, Stowers SJ, Miller EC, Anderson MW, Miller JA (1986) Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F~ mouse. Proc Natl Acad Sci USA 83:5825-5829 Wittig B, Dorbic T, Rich A (1991) Transcription is associated with Z-DNA formation in metabolically active permeabilized mammalian cell nuclei. Proc Natl Acad Sci USA 88:22592263 Wolfe KH, Sharp PM, Li W-H (1989) Mutation rates differ among regions of the mammalian genome. Nature 337:283285 Wood RD, Coverley D (1991) D N A excision repair in mammalian cell extracts. BioEssays 13:447-453 Wyman AR, Wolfe LB, Botstein D (1985) Propagation of some h u m a n DNA sequences in bacteriophage ~ vectors requires

180 mutant Escherichia coli hosts. Proc Natl Acad Sci USA 82: 2880-2884 Zannis-Hadjopoulos M, Kaufmann G, Wang S-S, Lechner RL, Karawya E, Hesse J, Martin R G (1985) Properties of some monkey DNA sequences obtained by a procedure that enriches for DNA replication origins. Mol Cell Biol 5:16211629 Zannis-Hadjopoulos M, Frappier L, Khoury M, Price GB (1988) Effect of anti-cruciform DNA monoclonal antibodies on DNA replication. EMBO J 7:1837-1844 Zhang S, Grosse F (1990) Accuracy of DNA primase. J Mol Biol 216:475-479 Zuckerkandl E (1976) Programs of gene action and progressive evolution. In: Goodman M, Tashian RE (eds) Molecular anthropology genes and proteins in the evolutionary ascent of the primates. Plenum, New York, pp 387-447 Zuckerkandl E (1985) Toward a molecular biology of predisposition to disease: the case of cancer. In: Huemer RP (ed)

The roots of molecular medicine. A tribute to Linus Pauling. WH Freeman, New York, pp. 1-27 Zuckerkandl E (1992) Revisiting junk DNA. J Mol Evol 34: 259-271 Zuckerkandl E, Pauling L (1965) Evolutionary divergence and convergence in proteins. In: Bryson V, Vogel HJ (eds) Evolving genes and proteins. Academic Press, New York, pp 97166 Zuckerkandl E, Derancourt J, Vogel H (1971) Mutational trends and random processes in the evolution of informational macromolecules. J Mol Biol 59:473--490 Zuckerkandl E, Latter G, Jurka J (1989) Maintenance of function without selection: Alu sequences as "cheap genes." J Mol Evol 29:504-512

Received October 17, 1991/Revised January 17, 1992/Accepted February 25, 1992