Cis and trans mechanisms Jean-Michel University

of North

Carolina,

of DNA repair

H. Vos

Chapel

Hill, North

Carolina,

USA

DNA repair is essential for genetic stability and variability. Remarkable advances in the understanding of DNA repair by the molecular analysis of the substrate (gene repair) or the enzyme (repair genes), emphasize evolutionary conservation. Recent progress also stresses the interaction(s) between DNA repair and numerous other cellular metabolic processes, including non-nuclear and/or non-genetic responses.

Current

Opinion

in Cell

Biology

Introduction

4:385-395

to a prolific and sometimes conflicting literature in this field. Although this analysis has been applied mostly to the repair of ultraviolet (LJV)-induced cyclobutyl pyrimidine dimers (CPDs), it has generated four tentative and general rules. First, actively transcribed genes can be preferentially repaired. Second, preferential repair of some expressed genes results from selective repair of their transcribed strand. Third, the DNA repair appears to be evolutionarily conserved from bacteria to humans (although not yet described for viruses). Finally, DNA repair is important, and this is underlined by its apparent deficiency in the neurogenerative disorder Cockayne syndrome (CS). One way to delineate the nature of a repair pathway is fo identify genes, species or mutants that deviate from it (the so-called genetic approach); another way is to develop an in tv’fro assay for preferential repair using cell extracts (the so-called biochemical approach).

The intrinsic chemical instabilitv of DNA, the spontaneous generation of DNA modibers by cellular metaholism and the continuous presence of DNA-damaging agents in the environment are all responsible for COW stanr alterations in genetic material ranging from that of simple viruses to complex multicellular organisms. To respond to such deleterious insults, living creatures have evolved the capaciv to remove or tolerate lesions in their DNA [ 11. The scope of this review is to provide the reader with an overview of the 1991 literature on DNA repair. The only way to limit this review to a reasonable length has been to focus on selected topics, and often to provide a particular viewpoint. This cannot be done without appearing arbitrary and neglecting many good papers. For pre\ious years, I refer the reader throughout to other excellent and comprehensive reviews. Bearing in mind that the aim of this issue is to gather information on the diversified nuclear metabolic processes, I have attempted to emphasize the continuously strengthening relationship between DNA repair and other major DNA transactions such as transcription, replication and recombination. Mutagenesis and prokaryotic DNA repair studies have been cited in direct connection with DNA repair in eukayotes. The conjunction of DNA repair and molecular bioloa has led to the expansion of different aspects of this rapidly evolving field, helped 1~~ the development of techniques both to study repair at the gene-specitic level and to isolate genes involved in repair. Gene

1992,

Coupling

of repair

and

transcription

in bacteria

There are two broadly defined models that could explain selective repair of the transcribed strand: a passive ‘structural’ model, whereby a specific conformational change in the transcribed DNA template induced by the stalled RNA polymerase, with or without a partially hybridized RNA, is the main signal; or an active ‘coupling factor’ model, in which a defined gene product triggers the response through specific protein-protein interactions after detection of the transcriptional block. Two papers reported by Sancar and coworkers [4,5**] support the second model. On the one hand, an in l&-o assay with concommitant transcription and ulpr-mediated excision repair allowed the biochemical identilication of transcription-repair coupling factor in (TRCF) f%ccbet-ichin coli extracts [4] ; this factor stimulates by several-fold the selective removal of bulky lesions such as CPD and psoralen monadducts (PMAs) from the transcribed strand of a highly transcribed gene. TRCF has

repair

it has long been realized that DNA repair in eukaryotes varies with genomic location [ 2,3], recent systematic analysis of repair at the gene-specific level has led

tithough

Abbreviations AT-ataxia

telangiectasia; DDI-DNA

CP[tcyclobutyl damage

pyrimidine dimers; CSCockayne syndrome; DDB-DNA inducible; ERCC-excislon-repair cross-species complementation;

PMA-psoralen monoadduct; TRCF-transcription-repair

@

pol-polymerase; coupling factor;

Current

Biology

scid-severe UV-ultraviolet;

Ltd

ISSN

combined XP-xeroderma

0955-0674

damage

binding;

immunodeticiency; pigmentosum. 385

386

Nucleus

,and gene expression

a molecular mass of 121 kD and appears to be, as yet, an unidentified E. coli protein (A. Sancar, personal communication). On the other hand, a careful search of the literature led the same authors to the previously uncharacterized mutator phenotype mfd [ 5**]. They demonstrated that although extracts from mfd-bacteria are deficient in selective repair of the transcribed strand, a fraction enriched in TRCF activity can restore such selective repair. Thus, mfd appears to be the most likely candidate gene for encoding TRCF, which could drive the uvr ABC excinuclease for selective repair of the transcribed strand, possibly by direct protein-protein interactions with the transcription and/or the repair complexes. Remarkably, elegant work by Bockrath and coworkers 15 years ago had led them to propose that “mfd is a unique process involving excision repair of premutational lesions located only in the transcribed strand of DNA” (see [5**] >. The bacterial mfdgene may be equivalent to the defective human gene in CS, which apparently has lost the ability for strand-specific repair of expressed genes (reviewed in [6]).

‘Domain’

versus ‘strand’

repair

in yeast

Recent experiments with yeast have revealed the complexity in transcription-repair coupling. Smerdon (see [2]> used a method of site-specific detection of CPD to follow the repair of individual CPD on an engineered episome in Succharomlces cerezvsiae, and demonstrated selective strand repair-in the transcribed regions of the plasmid. Recently, they found that stable nucleosomes could override such tight coupling between transcription and repair at low, but not at high, transcription rates [7]. In contrast, Terleth et al. [3] reported that preferential repair of CPD on the active mating type M4T gene did not derive from strand-specific repair and was maintained in the absence of transcription. These apparently conflicting results point to an intriguing possibility: cis elements may control preferential repair on both strands, e.g. by chromosomal position, while trualzs acting factors, such as TCRF, may control selective strand repair.

tack of preferential

repair

in Drosophila

Preferential repair has not been observed in Kc cells from the fruit& Drosophilu mehnogaster [8]; UVinduced CPD were removed at the same rate from the active gurt, the inactive notch and white genes, or from the genome overall, and no strand-specific repair was detected. This may reveal an evolutionary limitation of preferential repair to certain eukaryotic phyla such as fungae and mammals. If so, such a division may derive from basic differences in chromatin organization between insects, such as D. mehnogaster, and other eukaryotes; for example, only 10% of the D. meianogaster genome is heterochromatin, whereas in mammalian cells this figure is much higher. Thus, slow repair may be limited to the heterochromatic regions [9]. However, as immortalized embryonic cells were used, it is possible that preferential repair only becomes operative at a later stage of development or that it is lost during immortalization of this cell line.

‘Repair

cold spots’ in mammals

Using the photocatalyzed reaction of psoralen as a model of DNA lesion, and a sensitive assay to compare directly DNA crosslinking levels between genes, Vos and Wauthier [lo**] unexpectedly found that preferential repair did not occur in rRNA genes in either human or Chinese hamster cells. In addition, they observed that although active rRNA transcription increased the level of psoralen modification by several-fold in human-mouse hybrids, repair was equally inefficient on active and inactive rRNA genes. Since mammalian rRNA genes are transcribed by RNA polymerase (pal) I, preferential repair may be restricted to genes transcribed by RNA pol II. The high copy number of rRNA genes and RNA pol III-transcribed genes may not necessitate their preferential repair. Mutagenesis of these highly conserved multicopy genes could be prevented by mismatch-repair/gene-conversion mechanism(s). The above study indicates that repair of rRNA genes is remarkably inefficient, i.e. below the repair rate of the genome overall [ 10°*,l 11. Defective repair of bulky DNA adducts has been reported on the silent repeated centromeric a-region from monkey cells [ 121. I propose that these ‘repair cold spots’ reflect the existence of a ‘DNA repair silencer’, e.g. c&mediated repair inhibition. By analog to DNA replication, chromosomes could be topologically divided into defined genomic mits of repair, or ‘repairons’ [ 131. Strand-repair,

asymetric

replication

and mutagenesis

Strand-specific repair of DNA damage in active genes is also supported by recent studies that compare mutation spectra in repair-proficient and repair-deficient cells or in synchronized-repair proficient cells treated at different times in the cycle [6,1-i]. Whereas mutagenic lesions in repair-proficient cells were mostly~ located in the non-transcribed strand, such strand bias WLS inverted in repair-deficient cells with most mutagenic lesions located in the transcribed strand. Thus, repair-proficient cells remove the mutagenic lesions preferentially from the transcribed strands of active genes. Interestingly, the strand bias observed in repair-deficient cells could indicate different mutation rates on the leading and lagging strands in mammalian cells [6]. If this is true, strandbias mutation will depend on the relative orientation of transcription and replication, which can be tested with shuttle vectors (see [ 151). Strand-repair,

demethylation

and mismatch

repair

Preferential excision repair of DNA damage is reminiscent of two other strand-selective excision-repair mechanisms that both involve methylated DNA: mismatch repair and gene activation. In the bacterial system, mismatched bases are selectively removed from unmethylated strands of hemi-methylated DNA [ 161. Higher eukatyotic cells possess general mismatch-repair systems that may by functionally similar to the methyl-directed pathway of .E coli. Such a system appears to perform strand-specific repair of a heteroduplex from a site-specific single-strand break [ 16,171 and is also consistent with the recent identification of potential mismatch-recognition activities in mammalian cells [ 161. Intriguingly, an unrelated study has indicated the existence of a transient strand-specific DNA

Cis

demethylation process in mammalian cells that is associated with transcriptional activation of methylated silent genes [ 181. Its authors proposed that such strand-specific modification occurred “via an active pathway through excision repair and/or enzymatic demethylation”. It will be interesting to establish if any overlap exists in strand selectivity between excision repair, mismatch repair and transcriptional activation, particularly in eukaryotic cells.

Repair

genes

If the understanding of excision repair in prokaryotes as exemplifed by the uvrABC excinuclease [ 191 has reached adulthood, then the equivalent process in eukaryotes is still in its infancy (Fig. 1). In both yeast and mammalian cells, the molecular isolation and characterization of repair genes and gene products has been pursued using genetic strategies, such as DNA-mediated complementation of repair-deficient mutants (or syndromes), and biochemical strategies such ‘as protein purification from excision repair-competent cell extracts.

The

biochemical

route

DNA damage recognition Both constitutive and inducible DNA damage binding (DDB) activities operate on many different types of DNA lesions in mammalian cell extracts [20,21]. Cells from some patients with xeroderma pigmentosum (XP), an excision repair-deficient syndrome, appear not to have DDB activities [21,22]. As XP-E and XP-A patients have different genetic defects, it is probable that more than one protein is required for DNA damage recognition in human cells. Whether DDB factors are also directly involved in the latter steps of excision repair, or overlap with other DNA-repair processes, is not yet known.

DNA damage incision Wood, LindahI and colleagues (see [ 211) have developed an elegant experimental strategy to study excision repair using mammalian cell extracts Based upon the detection of in llitro repair synthesis on damaged plasmid DNA, it provides an extremely useful starting point for further characterization of repair proteins and has been used to show that repair occurs on various bulky DNA damage, is located at the site of damage and leads to removal of damage. An average repair patch size of 2540 nucleotides has been observed it? rlitro [ 231, and this would conlirm earlier in rliljo estimates [ 11. Recently, it has been shown that the DNA lesion is removed as a 27-29 oligomer in human cell extracts via an excision nuclease enzyme s)lstem, with endonucleolytic incision at 23 bases on the 5’ side and 6 bases on the 3’ side of the lesion [ 24.1. Apart from the difference in size of the released oligomer with the lesion, i.e. 12-13 bases in E. coli but 27-29 bases in human, such a pattern of DNA incision appears analogous to the bacterial uvrABC excinuclease [ 191. It still remains to be established whether in humans the two incisions on either side of the lesion are simultaneous or sequential,

and

trans

mechanisms

of DNA

reoair

Vos

Repair synthesis The role of identified cellular proteins on activities measured in crude cell extracts may be determined by testing the effect of specific neutralizing antibodies. Such ‘guess’ strategy was used to demonstrate the stimulation of excision repair by two human replication co-factors, the single-stranded DNA-binding protein, HSSB [23], and proliferating cell nuclear antigen (RD Wood, personal communication). Both studies appear to exclude DNA pol a and p from the step of repair synthesis in excision of bulky damage from human DNA, indicating that either DNA pol 6 and/or pal E is the main repair replication enzyme in humans [25]. In contrast, a role for DNA pol p in DNA semi-consemative replication has been proposed [26]. However, in vitro assays with purified components are required to establish protein functions fully. The recent extension of the human cell-free excision-repair system to rodent cell extracts (RD Wood, personal communication) and to yeast cell extracts (EC Friedberg, personal communication) should help the molecular identification of repair genes from various species. In vitro chromatin repair All the in rdtro studies discussed so far have used purified DNA as a template for repair. The extensive evidence for intmgenomic heterogenity of repair, particularly on eukaryotic chromosomes, points to the need for cell-free excision-repair systems that are active on DNA damage embeded in chromatin. A recent report that nucleotide excision repair by human cell extracts is suppressed in reconstituted nucleosomes may provide a means of isolating factors controlling recognition and access to DNA lesions in chromatin [ 271. The

genetic

route

Functional interspecies complementation of repair-deficient cell mutants by DNA transfection has proved a powerful approach to the cloning of eukaryotic repair genes. Sources of repair-deficient mutants have been provided from irz l+tt-o selected cell mutants, e.g. yeast RAD mutants [ 281 and rodent cell mutants [29], or from identified inherited human syndromes with hypersensitivity to DNA damaging agents, e.g. XP [ 11. The large number of genetically distinct complementation groups - there are at least 10 genes for excision repair of UV damage in eukavotic cells [28,29] - illustrates the biochemical and molecular complexity of the excision-repair process in eukaryotes. Since the first cloning of mammalian genes correcting a defect in excision repair of a rodent mutant (ERCC I) and a human syndrome (Xf?AC), several other human excision-repair genes have been isolated [30]. Tumor suppression and point mutations in a human repair gene Tanaka and colleagues ( [31*]; K Tanaka, personal communication) have identified 11 different mutations of the XPACgene in XP-A patients of diverse ethnic origin and varying disease severity. Nine point mutations consisted of three splicing mutations (3’ ends of intron 3, exon 3 and exon 4), four nonsense mutations (amino acids

387

388

Nucleus

alrd gene expression

. (a) Bulk

Disruption

repair

(b)

Domain

repair

G

XP-A, XP-E?

Binding

XP-A,XP-E?

XP-A,XP-E!

+I a

3-l

Unwinding

Recognition

Incision

Removal

Synthesis

Ligation

Packaging

0 @

A

Nucleosome DNA

(silent)

damage

Accessibility

0

Nucleosome Excision

factor

DNA

(poised)

(

nuclease polymerase

+ 6/c

0

RNA

polymerase

DNA

helicase

Transcription-repair

II

DDBP

v 0 coupling

iactor

SSBP

Cis and trans mechanisms

116, 207, 211 and 228) and two m&sense mutations (CyslO8 to Phe and His244 to Arg). Two small deletions creating frameshifts in exon 3 were identified as a single-base deletion (nucleotide 374) and a five-base deletion (nucleotide 349-353). Interestingly, mutations in the zinc-finger domain of the XPAC protein were observed in severely affected XP-A patients. These results strongly suggest that the seventy of clinical symptoms, especially neurological symptoms, depends on the residual activity of the truncated XPAC protein. This study confirms at the molecular level the fundamental role of a human repair gene in tumor suppression and is reminiscent of the analysis of inactivating mutations of the anti-oncogene ~5.3 [ 321 radiation

resistance

gene

The molecular complexity involved in the repair of radiation-induced damage is underlined by the existence of at least six genetically distinct complementation groups in rodent [33]. Thompson and colleagues [34*] reported the isolation of the first mammalian gene, the human XRCCI gene, involved in repairing DNA singlestrand breaks produced by ionizing radiation and alkylating agents. Such a repair pathway appears distinct from that which repairs DNA double-strand breaks. No significant sequence homology was found between XJKCI and other genes, including yeast RAD genes, so the biochemical function of the XKCl product remains unknown. The elucidation of such an X-ray repair pathway may help the study of radiation resistance in tumor cells and the development of improved radiation-based cancer therapy. DNA helicases and repair of active genes for excision-repair cross-species complementation

As is usual with the ‘reverse genetic’ approach, the elucidation of the precise biochemical functions of cloned repair genes has proved difficult. Sequence homology with proteins of known functions may indicate potential function, such as DNA binding for excision-repair crossspecies complementation (ERCCI-l/RADIO and XPAC and DNA helicases for ERCC3/XPBC/CSA and ERCC6/CSB [ 301. The recently cloned ERG2 gene has strong homology with yeast R4D3 [35] and has been shown to complement the Xp- D group (CA Weber and LH Thompson, personal communication; EC Friedberg, personal communication). Interestingly, the yeast I&4L?3 gene is known to be essential in uirlowith potential roles in replication, mismatch repair and recombination [ 281. In addition, the demonstration of DNA-DNA and DNA-RNA helicase activities using purified RAD3 protein with strand-

Vos

specificity [36*] may suggest a role for RAD~ in repair of active genes. If this is true, the XP group D may be deficient in preferential repair of active genes. Because of putative helicase activities it will be interesting to search for possible interactions between the gene products from ERG2 (XPDUfGID3) and those of genes affected in different complementation groups from CS, e.g. ERCC 3 ( CTclIxpB) or ERG6 (CSB), particularly for the repair of active genes. For example, such pleiotropy of helicases may be required for the repair of different genomic regions or for different steps in excision repair of active genes. Replication

A high-energy

of DNA reDair

and DNA damage

Unrepaired DNA damage interferes with the DNA replication machinery in dividing cells. Although replication of DNA lesions is widely documented in prokaryotes and eukaryotes, the mechanism(s) and fidelity of lesion tolerance processes are still poorly understood, particularly in eukaryotes. Such processes are known as post-replication repair, bypass replication or trans-lesion replication [ 11, although they may describe distinct mechanisms. Shortterm replication of DNA damage may help circumvent transcription blockage from bulky lesions located on active genes [37]. Long-term replication will lead to various genetic changes, ranging from simple point mutations to complex chromosomal rearrangements. By analogy to prokaryotes, one would expect the coexistence of distinct error-prone and error-free replication pathways in eukaryotes. Although both genetic and biochemical strategies are being used to unravel the mechanism(s) of DNA damage replication, progress has been slow. For example, recent years have seen major advances in our understanding of replication of undamaged eukaryotic DNA as a result, in particular, of the development of in L&-o replication systems. In contrast, the efficient bypass replication observed in intact cells [37] has yet to be obtained in cell-free systems. Although such systems have been used extensively for mutagenesis analysis of various mammalian DNA polymerases [38], the studies have relied on extremely sensitive mutagenic assays. Thus, there is still a need for efficient, eukaryotic-based, in vitro bypass replication systems. DNA replication,

protein

modification

and yeast meiosis

Mutants affected by the replication of damaged DNA have been described in yeast, e.g. at least six distinct genes in Succhuron~yces cerezdae [ 281. In particular, the yeast

Fig. 1. A schematic model for eukaryotic excision repair of damage DNA with potential defects in human syndromes. Different levels of repair correspond to (a) silent regions, (b) functionally poised regions and fc) transcribed regions. Bulk repair requires disruption of the nucleosomes in order to obtain access to the DNA. Domain repair may depend upon an accessibility factor [641. Strand repair may involve a transcription-repair coupling factor and DNA helicase activity f651. Recognition and access to damaged DNA is followed by specific DNA-protein binding the lesion encoding 27-29 amino acids is removed by a putative excision nuclease and the single-strand gap complexed with the single-strand binding protein (SSBP) is filled by DNA polymerase h/E and sealed by DNA ligasefs). XP, xeroderma pigmentosum; CS, Cockayne syndrome; DDBP, DNA damage binding protein.

389

390

Nucleus

atid

gene

expression

. RADGgene is known to play a central role in post-replication repair, damage-induced mutagenesis and sporulation by encoding a ubiquitin-conjugating (E2) enzyme that may be involved in histone degradation and modulation of chromatin structure [ 281. Its evolutionary conservation from yeast to human [39*] indicates its basic function(s) in eukaryotes. Genetic analysis suggests that the ubiquitination activity of the RAD6 protein is essential for all its functions; thus, its repair/mutagenesis functions appear to be mediated by the ubiquidnation and degradation of non-histone chromatin proteins or other repair proteins [40]. Proteins that may be involved in directing RAD6 to the damaged site or may be a biological substrate of RAD6 could include those encoded by genes in the R4DGepistasis group, e.g. the putative DNA-binding RADl8 protein, or the REVSfamily [ 28,401. Intriguingly, at least two genes in this group, RADGand RAD18, appear to be co-regulated by UV in yeast [41]. The multifunctional nature of this protein underscores the complexic) of the mechanism(s) of replication of DNA damage in eukaryotes.

Gene-specific pigmentosum

bypass variant

replication

defect

in xeroderma

Several human syndromes, such as XP variant (XPV), CS and ataxia telangiectasia (AT), present *ananomalous pattern of replication after DNA damage [ 11. Apart from the recent advances made with CS (see above), it has been difficult to pinpoint the exact defect in these syndromes. XPV is a particularly attractive candidate mutant with a defect in bypass replication, as suggested by recent mutational studies [ 421. Using an assay to measure repair and replication of psoralen adducts at the genespecific level, it was previously reported that PMA are efficiently bypassed in cells, indicating high persistence of such adducts on both the leading and lagging DNA strands [36*,43]. XPV cells have recently been shown to be partially defective in bypass replication of PMA [ 441. As approximately half the intrastrand adducts were bypassed during replication in XPV cells, compared with normal cells, it was proposed that the XPV defect ma) be strand-specific, i.e. bypass of adducts occurs on either the leading or the lagging strand. Furthermore, as the low level of replication of psoralen interstrand crosslinks that usually occurs in normal cells [36*,43] was not observed in XPV cells, replication of DNA damage in human cells may involve strand-specific homologous recombination (Fig. 2).

Recombination

and DNA damage

As DNA recombination has been the focus of many excellent reviews, including one by Haber (this issue, pp 401-4121, 1 will only present recent advances in the understanding of recombination in relation to the processing of DNA damage. The role of homologous recombination in DNA damage processing may be especially important for the repair of bi-strand DNA damage, such as an interstrand crosslink, a double-strand break, closely opposed or gap opposed intrastrand damages.

Bacterial

resolution

of Holliday

structure

Much of our current understanding of homologous recombination is derived from in tv’tro studies of prokaryotic proteins [45]. West and colleagues [46**] have recently shown that DNA recombination can be reconstituted irl Llitro using two purifed enzymes from E. co/i: recA, which promotes the pairing of homologous DNA molecules and catalyzes strand exchange leading to the formation of heteroduplex DNA, and the newly identified RuvC protein, which resolves the intermediates by specific endonucleolytic cleavage to produce recombinant molecules. The role of both enzymes in recombinational repair of DNA damage has been well established through the hypersensitivity of recA or RrtzC mutants to a plethora of DNA-damaging agents. It is not yet known if any of the myeast mutants deficient in recombinational repair [28] are affected in recA or RuvClike activities. Antirecombination

and

homologous

DNA

Evidence is emerging that specific DNA pathways may prevent, rather than promote, DNA recombination. The elegant work of Radman and colleagues [47] shows that a mismatch repair system may inhibit recombination between partially homologous sequences and provide a functional barrier to interspecies recombination. This study directly implicated the bacterial mur-dependent mismatch repair system [ 161 as anti-recombination function by demonstrating successful genetic exchange between ??lztl( 1 bacteria with 20% sequence divergence. Mismatch repair may also suppress intrachromosomal recombination between highly repeated elements, thus stabilizing the mammalian genome despite the presence of millions of repeats [47,481, However, the molecular basis of anti-recombination has not been established. The failure of recombination may be explained, at least in part, by the following processes: reversal of the exchange process by a mismatch-sensing helicase; mismatch-dependent incision of the invading strand by an endonuclease; or excision of the invading non-homologous strand by an exonuclease. Intriguingly, strand-specific mismatch repair in human cell extracts has been recently described [ 16,171. If and how DNA damage and/or excision repair may interact with the processes of homoeologous recombination and mismatch repair has yet to be explained. Regulation

of DNA

repair

The paradigm repair system regulated by exposure to DNA-damaging treatment is the bacterial SOS response [ 11; approximately 20 genes involved in DNA repair, mutagenesis and recombination are induced by UV Mdiation or chemical agents. Damage by alkylating agents or oxidative stress induces other well characterized regulons in bacteria [ 1,491. In yeast, it has been estimated that there may be up to 80 DNA damage inducible (DDI) genes, including at least 35 R4D genes involved in excision repair, mutagenesis, post-replication repair or recombination [ 281. Although a large number of DDI cDNA clones have recently been isolated from mammalian cells, only a few of their products have recently been identified [SO]. Activation of the UV response leads to the induc-

Cis

and

trans

mechanisms

of DNA

reDair

Vos

Replication

Replication

Incision

Bypass

Recombination

DSB

Repair

Allele

recomhinaton/ repair

Allele-

C -

Er w

Fig. 2. A hypothetical model of error-free bypass replicatron of rntrastrand and interstrand DNA damage (0) based on general recombinatron. Replicatron forks move along homologous regions of alleles with the upper and lower strands as leading and lagging strands, respectively. (a) Bypass replicatron of intrastrand damage, e.g. psoralen monoadduct, on the lagging strand via the repair of a single-strand gap left by an rncomplete Okazaki fragment using the parental lagging strand as a template for recombination. (b) Bypass replication of intrastrand damage on the leading strand via the blocked leading strand switching template and using the new complete lagging strand instead. The new strands then disassociate to reanneal with their original template strands. fc) Bypass replication of interstrand damage, e.g. psoralen crosslink, via the repair of a transient double-strand break. Another duplex allele (not shown) is used as the template for the recombrnation-mediated double-strand break reparr.

391

392

Nucleus

and

gene

expression

. tion of some gene products, such as methallothioneins, plasminogen activator and collagenase, whose role in DNA repair and protection of the cell against genotoxic treatment is not obvious. Also unclear is the overlap of the pattern of gene induction between different adverse agents, such as heat shock, toxic heavy-metals, UV, phorbol esters, and positive signals, such as growth factors or hormones [ 501.

of other cellular factors [ 52,53*]. Thus, it will be of interest to identify the primary targets for redox-mediated inducible DNA repair responses in eukaryotic cells. Unfortunately, it is likely that multiple initial triggering factors exist, depending on the cell-type and the DNA-damaging agent, and including post-translational modifications such as phosphorylation (see above) or poly-ADP-ribosylation [541.

Double-strand cycle in yeast

Repair deficiency, syndromes

breaks,

protein

phosphorylation

and

cell

Two studies based upon the eukalyotic site-specific HO cleavage system have identified tram-acting cell-cycle regulatory signals for DNA repair in budding yeast. Hoekstra el al [ 51.1 isolated a new r&like gene, HRR25, that belongs to a protein kinase superfamily. As HRR-5 is required for normal cellular growth, nuclear segregation, repair of double-strand breaks and meiosis, and has sequence homology to protein kinase genes involved in cell cycle progression and DNA repair, such as CDC7, the HRR25 kinase can be placed in the functional categoly of yeast kinases associated with DNA metabolism. It will be interesting to isolate analogous mammalian Hm5 gene(s). In an elegant study, Resnick and colleagues (M Resnick, personal communication) demonstrated that a single double-strand break on a non-essential plasmid is sufficient to induce cell cycle arrest and lethality in yeast. Surprisingly, part of the arrest was RAD9-independent, suggesting that other DNA damage-dependent cell cycle regulatory processes are involved. This system provides a powerful means of identifying the nature of the molecular signal(s) triggering cell cycle arrest and gene activation after DNA damage and may also function in mammalian cells. Stress signal, in mammals

protein

oxidation

and

transcription

factors

It would be particularly interesting to identify the intracellular initiator molecule(s) triggering the cascade of induced and repressed DNA repair events. A number of transcriptional regulators are induced early after DNA damage [50]. Intriguingly, two recent studies suggest redox regulation of transcriptional factors [52,53*]. In the nuclear AF-1 complex, oxidation of a single cysteine residue in the DNA-binding domains of the Jun and Fos proteins directly regulated DNA-binding of the Jun-Fos heterodimer [ 53.1. This is reminiscent of the bacterial transcriptional regulator); protein OxyR, which regulates gene expression in response to oxidative stress via its redox state [49]. In both cases, activation through oxidation does not involve an intramolecular or intermolecular disuffide bond: it may involve either a tightly-bound redox-active metal or reversible o.xidation of the critical cysteine. In contrast, activation of NFxB probably involves inactivation of its cytoplasmic scavenger IlcB via either irreversible oxidation or selective proteolytic degradation [ 53’1. Thus, paradoxically, &ox-mediated modification of proteins, instead of DNA, may be a general mechanism for DNA damage inducible responses. However, although the bacterial OxyR can be directly activated in vitro by oxidation, activation of mammalian factors AI-1 and NFxB may require modifications

signal

transduction

and

human

Recent data suggest that the primary defects in AT may lie in ‘master’ regulatory genes whose products control the expression of multiple pathways, including DNA repair [55). 1 propose that this may also apply to other DNA repair deficiency s)~dromes with complex features, such as Bloom’s s\ndrome and Fanconi’s anemia. The multifaceted clinical phenotypes of the diseases, the i)z l*ifro segregation of various molecular defects, the correction or transmission of the defects through diffusible factors, the abnormal production of intracellular or extracellular proteins, and the well established cell cycle abnormalities all suggest that these syndromes may be tiected by mechanism(s) interacting with membrane signalling and transduction circuitry and/or the pleiotropic stress responses described above. A5 these processes play a fundamental role in the control of normal cell growth and development, understanding the molecular defects of these diseases may help to shed light on the nature of such putative regulatoqr networks, in addition to DNA repair processes per se. Repair

in multicellular

organisms

Analysis of DNA repair in multicellular organisms is important for a number of reasons: first, to study the developmental regulation of DNA repair, such as differential expression of repair genes or differential chromatin accessibility to repair enzymes; second. to understand how DNA repair relates to complex and pathogenic multicellular processes such as cancer and aging; third, to establish inter-individual variation in DNA repair capacity for vatious tissues; and finally, to establish DNA repair deficient transgenic animals as models of human syndromes. Of particular interest are eukatyotic organisms with developmental and genetic attributes such as the fungus Neuro spora crussa, the slime mold Dichm-telillrn discoideum, the nematode Caenorbahditis elegnns, and the fruitfly D. nzelanogusfe?: As the description of DNA repair studies for these different organisms is beyond the scope of this review, the reader is referred to other related excellent articles [ 56,571. I will limit the discussion that follows to recent studies on rodents with spontaneous or engineered DNA repair mutants/variants. Double-strand immunodeficiency

breaks,

DNA

recombination

and

Following the report of hypersensitivit), to ionizing rddiations of cells from mice with the se\.ere combined immunodeficiency mutation (s&Y). two groups have now demonstrated that the defect specifically relates to the

Cis and tram mechanisms

repair of double-strand DNA breaks [58,59]. Such a step could involve proteins that hold the ends together, specific endonuclease and/or exonucleases to trim modtied residues or polymerase(s) and ligase(s) to close the repaired strands. Although there are numerous mutant rodent cell lines that are hypersensitive to ionizing radiation [33], the scid mutation is almost unique in conferring radiosensitivity to the whole animal: the AT mutation in humans is the only other example. The scid defect clearly establishes the overlap between different DNA modification pathways in rodents, such as ubiquitous repair of random double-strand DNA breaks and tissue- and site-specific recombination. Other specific DNA transaction(s) may also incorporate the use of DNA repair proteins. Whether such a possibility also applies to immuno-compromised humans with an inherited defect in DNA repair, such as AT, CS and some XP cases, remains to be established. Mutagenic transgenic

lesions, mouse

bacterial

repair

enzyme

and

The advent of transgenic animals has made possible the study of single gene products ilz ldlo in a stable genetic background [6Ol. The transgenic approach can be used to overexpress or, conversely, inactivate a specific protein. Although progress has been made in the construction of ERCCs-null mice (K Tanaka, personal communication; LH Thompson, personal communication), the first report of transgenic repair-affected mice is based on the overexpression of a bacterial repair enzyme (611. Gerson and colleagues [61] have demonstrated that transgenie mice expressing a tissue- and diet-regulated E. cofi 06.aikylguanine-DNA alkyltransferase (r&l) gene have enhanced repair of 06.methylguanine DNA adducts in their liver. Since these adducts are known to be mutagenic and are suspected to be carcinogens, these mouse strains, as well as future null-transgenes for endogenous repair enzymes, will be useful in long-term chemical carcinogenesis studies.

may help to shed light on poorly understood processes such as memory and aging. Acknowledgements This paper is dedicated to Dr. Philip Hanawalt for his 60th birthday anniversary. I am grateful to the many colleagues cited in this review for communicating unpublished data and I apologize to many others for not being able to cite aU the relevant work. I thank the members of my laboratory for helpful discussions and Amy Lowery for excellent secretarial assistance. Work from the author’s laboratory cited above was supported by grants from the NCI (1.ROlCA51096), DOE (DEFGO59lER611350), the Life & Health Insurance Medical Re. search Fund and the Elsa U. Pardee Foundation. The author is the recipient of a Basil O’Connor Starter Scholar Research Award from the March of Dimes Birth Defects Foundation (5-813) and of an American Cancer Society Junior Faculty Award (JFRA330).

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