Replication
origins,
factors and attachment
sites
Susan M. Gasser Swiss Institute
for Experimental
Cancer
Research,
Lausanne,
Switzerland
The initiation of eukaryotic DNA synthesis occurs at specific sites determined by both cis- and tram-acting elements. Here I review advances in the characterization of yeast origins, origin-binding proteins and the relationship of DNA replication to nuclear substructure in yeast.
Current
Opinion
in Cell
Introduction Our ability to reconstitute the cellular machinery for DNA replication in z&-o has progressed enormously in the past few years. Replication of simian virus 40 DNA can be reconstituted completely in a soluble reaction containing purified factors of the cellular replication machinery and the virally encoded initiator protein, large T antigen [I**]. Much of what we have learned, including the regulation of the system through phosphorylation and dephosphoryiation events, is directly applicable to genomic replication. However, the most important control point in genomic DNA synthesis - its initiation -will remain unexplained until the genomic sites of initiation and the proteins that recognize them are fully characterized. In the following pages I present recent advances in our understanding of the sequences required for initiation of DNA replication at yeast autonomously replicating sequence (ARS) elements, the proteins that bind origins, and some ultrastructural aspects of replication origins.
The yeast and ARS consensus AR.5elements were first isolated from yeast genomic DNA and from the endogenous 2micron circle because of their ability to increase the transformation efficiency of yeast integrative plasmids (reviewed in [ 2,3]). ARS elements allow the autonomous replication of large numbers of plasmid.s, with an unstable mitotic phenotype. The replication of ARS plasmids, like that of genomic DNA, requires entry into S-phase and initiation occurs only once per cell cycle. Proof that initiation occurs at or near ARS elements came from a two-dimensional gel electrophoresis system that allows separation of replication intermediates generated by the restriction digestion of replicating plasmids [4]. With this method, and a complementary neutral/alkaline system [ 51. ARSelements and genomic origins have been mapped on the ring-deriva-
Biology
1991,
3:407413
tive of chromosome III of Sacdaromyces cerevbiue. Five out of ten mapped ARS elements are used as chromosomal origins, and four others remain to be tested [2]. There are, however, no origins without ARS activity, and at least one ARS is necessary for maintenance of the circular chromosome III. All ARS elements contain one or more copies of a perfect or near-perfect match to an 11 bp A/T-rich sequence (WTITATRTTTW[W=AorT;R=AorG])thatisnecessary but not sufficient for ARS activity. In addition, A/Trich flanking DNA is required. The fact that single basepair mutations could abolish replicative activity suggested the importance of the ARS consensus [6]. However, single mismatches appear to be tolerated at many other ARS elements. To resolve this apparent contradiction, Van Houten and Newlon [7**] have mutated, to each of the three other possibilities, every nucleotide in the ARS consensus of a semisynthetic ARS element derived from the C2Gl ARS (now designated ARS307). These 33 point mutations were reintroduced into centromere-contaming vectors carrying the SUP11 and URA3 markers to allow quantitation of high-frequency transformation and plasmid-loss rate under non-selective conditions. Every nucleotide was found to be important for maximal ARS function, including, but to a lesser degree, the residue in position 12 and three bases located 5’ of the consensus (Fig. 1). Despite this strong demonstration of the significance of each nucleotide within the C2Gl ARS consensus, comparison with natural genomic ARS elements shows that the requirements are at least partially dependent on context. Among all sequenced ARS elements, only positions 7, 8, and 11 appear invariant. Thus, in certain contexts some, but not all, point mutations can be tolerated. Kipling and Kearsey [8**] have obtained results that underscore the importance of the ARS consensus using random mutagenesis to recover a replicating plasmid from non-replicating derivatives of an Ml3 vector. In two regions of the non-replicating Ml3 vector (not the region
Abbreviations ACBP-ARS
consensus-binding
protein;
ARS-autonomously SAR-scaffold
@ Current
Biology
replicating attached region.
sequence;
Ltd ISSN 0955*74
DHFR-dihydrofolate
reductase;
407
408
Nucleus
and gene expression
.
The ARS consensus-binding
protein
(a)
1
2
3
4
5
6
7
8
9
10
A
T
T
T
7
8
9
10
11
6) T 1
2
3
A 4
5
6
11
(c)
T
c
T
1
2
3
4
5
6
7
8
9
10
A/r
T
T
T
A
T
A/G
T
T
T
1
2
3
4
5
6
7
8
9
10
Two groups have identified a nuclear activity from S cerezvkiue that wiII recognize an oligonucleotide con taining the yeast ARS consensus [9**,10**]. In one cast the hydroxy radical footprint was determined on the H4 ARS element, the protein was purified, and the binding specificity of the 67 k~ protein for several genomic m elements and mutant ARS consenses was characterized [9-l. Although genetic evidence is lacking, the following observations are highly suggestive of a role in the initiation of DNA .synthesis.
11
(d) A/r 11
Fig. 1. A summary of important bases in the yeast autonomously replicating sequence (ARS) consensus sequence. Mutagenesis studies (a), reversion studies (b). and ARS consensus-binding protein (ACBP) studies (c) have contributed to the evaluation of the relative importance of the different residues in the yeast ARS consensus given here. The wild-type consensus sequence is indicated in (d) and the position of each residue is numbered. (a) For the C2Cl ARS, mutations at the following six positions within the core consensus resulted in an inactive ARS: from thymidlne (T) at positions 3, 8, 9 and 10; from adenosine (A) at position 5; and from guanine U to a pyrimidine at position 7. Mutations of the thymidines at positions 2 and 4 to adenosines weakened but did not eliminate ARS activity. Mutation to a base not inherent in the consenses sequence appeared to be tolerated in one instance only: thymidine to cytosine (C) at position 6. All other variation produced functional mutations (0). Cl, partially active mutation; n , inactive mutation. (b) Reversion studies in the Ml3 vector show that, for functional ARS consensus, six out of 11 bases are invariably wild type. These are identical to the invariant bases described in (a) and are given here in bold type. (c) Interaction of ACBP with. the wild-type consensus is greatly reduced by point mutations (given in bold type) at positions 3, 7 and 10. There is one permitted mutation (encircled) at position 5 where thymidine replaces the consensus adenosine. Intriguingly, this replacement is the only alternative base found at position 5 in naturally occurring ARS consenses: ARS121, ARS305, and ARS309 all contain thymidine at position 5.
containing a mutant yeast ARS element) the point mutations that confer AR.5activity were found to improve 9/l 1 near matches to the ARS consensus to lo/11 matches. Each mutation occurred in a region containing multiple 9/l 1 matches. The conserved positions among these ‘spontaneous’ ARS consenses are compared with the results of Van Houten and NewIon in Fig. 1. Remarkably, many of the conserved nucleotides common to these nvo studies are also conserved in the binding sites mapped for the AR.5 consensus-binding protein (ACBP) isolated by Hofmann and myself (9*=]. The consenlation of the AR.5 consensus argues for a consensus-binding protein, which would in some sense serve as the cellular analogue of T antigen or the bacterial DnaA initiation protein.
Footprint analysis irz llitro showed asymmetric protection of the 11 bp ARS consensus and adjacent sequences at the H4 ARS. A protein in the yeast scaffold extract bound specifically to the double-stranded form of a 22 bp oIigonucleotide containing the consensus and binds with higher af%nity to the T-rich, but not the A-rich, single strand of the same sequence. An excess of EscberidJiu coli single-strand DNA did not compete for the binding, whereas it competed eficiently for a major non-specific single-strand binding activity in the nuclear extract. The factor wa purified to apparent homogeneity as a 67kD polypeptide, ADBP, that retains the described DNA-binding properties, even after renaturation from a sodium dodecylsulphate gel. Apart from the oIigonucleotide, ACBP binds singlestranded fragments containing the perfect ARS consensus from four genomic ARS elements. Sequence specificity could be shown by quantifying the ability of the wild-type ARS consensus to compete for the binding of ollgonucleotides containing point mutations in the 11 bp ARS consensus within the semisynthetic C2Gl AR.5 element from the study by Campbell and Newlon [ 21. Three of the four point mutations that were tested significant11 reduced the affinity of the AR!%binding factor for the ARS consensus, while one (an A-T mismatch) appeared to enhance the affinity of ACBP for the consensus (Fig. 1). In addition, the affinity of ACBP for the ARS consensus appears to be stimulated by the presence of either of the leading-strand polymerases from yeast (6 or E) but not by DNA polymerase a or topoisomerase II. Unlike T antigen, ACBP has no detectable ATP-binding or helicase activity in its purified state (JFX Hofmann and SM Gasser, unpublished data). The significant variation of binding affinities due to single nucleotide changes demonstrates that the ACBP-DNA interaction is sequence specific. Within the range of its specificity, however, are certain 9/11 matches to the ARS consensus in single-stranded form. Near-match binding sites have been mapped in the 3’.flanking domains of the genomic ARSl and H4 ARS elements, and in each case the 9/11 binding site is on the opposite strand 3’ of the perfect consensus. At the H4 ARS, the two ACBP binding sites correlate with the boundaries of the minimal-functional AR.5element determined by deletion analysis [ 121. A model is presented in Fig. 2 that suggests a role for
Replication
the 3’ orientation of binding sites in the initiation of DNA synthesis.
(a) Recognition
and targeting
m (b) Helicase-mediated
unwinding
tc) Targeting polymerases leading-strand
of the 3’ element
for bidirectional synthesis
Fig. 2. Putatrve roles for an ARS consensus binding protein (ACBP) and the 3’ flank in the initiation of DNA replication. It is proposed that the autonomously replicating sequence (AR9 binding factor (represented by circles) first recognizes the double-strand ARS consensus and targets an associated helicase to the origin (a). Local unwinding in the 3’ DNA unwinding element (DUE) 1131 is Indicated in (b). ACBP should then recognize the T-rich strand of the perfect consensus and of the 3’ near-match to the consensus. It IS generally thought tin analogy to bacterial and viral systems) that synthesis starts through extension of an RNA primer by the DNA polymeraseu-primase complex. A transition to the leading strand polymerase ll**l, might then take place at the ARS consensus and its 3’ near match, through interaction of the leading strand polymerases 6 or E (~016, polo) with ACBP (c). The leading strand synthesis is indicated by the black arrows pointing in a 5’ to 3’ direction. Bidirectional replication may require two ACBP binding sites with 3’ orientation to each other.
origins,
The 3’ flanking
factors
and attachment
sites Gasser
domain
The observations on ACBP binding sites, and extensive deletion and mutagenesis studies, contribute to the two models put forth to explain the role of the DNA 3’ of the T-rich ARS consensus, a region that complements the ARSconsensus to provide replicative activity on plasmids. It has been shown that the 3’ region is hypersensitive to single-strand-specific endonucleases in negatively supercoiled plasmid DNA [3,13]. It would thus be a region of preferred unwinding in the presence of a helicase, and could serve as an entry site for the replication machinery. Umek and Kowalski [14*] have shown in viva that thermal energy (growth at 30°C as opposed to 23°C) can suppress mutations in the 3’ flanking region that decrease its propensity to unwind. This confirms that the double helix must unwind for origin hmction in zklo. The study of unwinding elements has been extended to the E. coli oriC, where three tandem repeats of a highly conserved 13-mer were suggested to serve as protein-binding sites and as the initial site of helix unwinding. The leftmost 13.mer can be replaced with another helically unstable sequence that contains only four matches to the consensus. This suggests that the potential for unwinding is more important than its sequence for one of the 13.mer repeats [15-l. On the other hand, a 13.mer-specific DNA-binding protein has recently been purified [ 16**]. This suggests that the repeated 13-mers serve both as protein-binding sites and as an easily unwound domain. A similar explanation is likely to be true for the yeast ARS 3’ flanking region. It has been suggested [ 171, that the essential contribution of the 3’ flank was multiple nearmatches to the ARS consensus, usually in an inverted onentation, which would provide binding sites for the then putative ARS-binding factor. The reversion studies on the Ml3 plasmid appear to support this idea, as mutagenesis created AR.5elements where multiple near matches were available on the opposite strand [ES**].Moreover, the nine genomic ARS elements presented by Palzkill and Newlon [ 171 and the telomeric ARS 121, studied by Walker et al. [ 1I**], all contain a 3’.flanking region with 9/l 1 matches to the ARS consensus inverted with respect to the T-rich strand of the major consensus site. In addition to preserving inverted ARS consenses, all these regions show sensitivity to single-strand-specific nucleases, indicative of an easily unwound domain. In some cases, linker mutagenesis and multiple point mutations in these near-perfect consenses seem to question the importance of the 3’ region as a protein-recognition site, at least in plasmid replication. At the telomeric ARS, mutagenesis of all three near matches decreases AR.5efhciency IO-fold but does not eliminate it [ 11 l *I At the H4 ARS, T-A mutations that maintain the propensity of the region to unwind, but at the same time destroy the nearmatch consenses, are still functional for plasmid replication [ 120*]. There are two possible explanations that remain consistent with a dual role for the 3’ flank: either the downstream binding sites are necessary only for the initiation of bidirectional DNA synthesis, such as one finds in
409
410
Nucleus
and gene expression
the chromosome (Fig. 21, while unidirection’al synthesis is sufficient for plasmid maintenance, or the run of seven Ts (or other 250 bp) of ‘SAR-DNA’ for efficient and detectable interaction with scaffolds, Within the ftz SAR, the ability to bind the scatTold in vitro correlated with enhanced plasmid replication, but was not a strict prerequisite for it 123’1.
Scaffold attachment may stimulate ARS activity in several ways. The sctiold-binding sequence may localize the ongin to a subcompartment of the nucleus that is rich in the factors and enzymes necessary for the initiation of DNA synthesis. In view of the AT-rich character of the SAR DNA, it may also coincide with the unwinding element discussed above, facilitating the opening of the double helix at the origin for entry of primase and polymerase activities, although scaffold attachment itself is not dependent on unwound or single-stranded DNA (T Falquet, personal communication). Finally, the SAR may provide repeated binding sites for accumulation of a specific initiation factor, such as ACBP, which itself fractionates with the scaffold fraction. Kiis et al. [24*] have also examined the scaffold binding capacity of AT-rich artificial polymers, and suggested that the oligo dT sequences and narrow minor-groove structure is the critical factor for scaffold interaction. They showed that distamycin, a drug that interacts specifically with a narrow minor-groove structure, competes efficiently for &AR-scaffold interaction. Moreover, the checkered dA,,Til polymer, which forms a rigid helix with a typically narrow minor groove, showed high affinity for the scaffold, provided it was sufficiently long ( > 160 bp). The same structural preference could be observed in the cooperative interaction of topoisomerase Il with SARs and artificial sequences in vitro [ 25.1. Studies on human and chicken &AR-scaffold interactions have come to similar conclusions [26*,27].
In summary, SARs and ARS elements share multiple structural properties. Eckdahl and Anderson [28] have pointed out that dA or dT stretches of threesix nucleotides in length, occurring at 10-11 bp intervals (resulting in bent DNA) and at 6-8 bp intervals (resulting in a rigid structure), are statistically enriched in yeast ARS elements and prokaryotic origins. These are identical with the sequences that characterize scaffold binding sites. Two important questions remain: whether the correlation between origins and scaffold attachment holds in
Replication
higher eukaryotes, and whether the SARs actually define the bases of DNA loops in intact cells.
A glance
at higher
eukaryote
origins
Sequence analysis of dihydrofolate reductase (DHPR) origin region reveals the presence of several long stretches of poly dT or dA sequence, stretches of alternating purine/pyrimidine pattern, and regions with distinctly bent character [29]. Several non-B-form DNA structures are favoured by the region, notably formation of triple helices and an unwound double helix. Some of the motifs found in the DHPR origin are also found in the two replication origins mapped by two-dimensional gel electrophoresis in the Drosophifiu chorion locus [30**] - in particular, the repetition of oligo dT stretches forming a region of bent DNA and near matches to the yeast ARS consensus. Intriguingly, a protein called RIPGO,purified from HeIa cell extracts, binds specifically to a repeated sequence element (ATT),, that occurs twice near the origin of bidirectional replication at DHPR, as well as recognizing the yeast ARS consensus sequence in z&-o [31-l (N Heintz, personal communication). It is not known whether these motifs are important for DNA replication at this locus, but the fact that RIPGOcopurihes with an ATP-dependent helicase activity is highly suggestive of a role in an initiation complex [31**]. The RIP60 protein itself enhances bending at its binding site, which is adjacent to a region of naturally bent DNA, a feature that often coincides with scaffold binding activity [24*,32*,33**]. Besides sequence homologies, there is experimental evidence that the origins of replication located 5’ of the
origins,
factors
and attachment
sites Casser
chick u-globin gene and 3’ of the Chinese hamster DHPR gene are both close to scaffold-attachment sites [34,35]. At least two origins of replication have been mapped within a 28 kb region downstream of the DHFR gene (see Hamlin et al., this issue pp 414-423), and the more gene-proximal initiation site appears to promote bidirectional replication [33**]. Several kilobases downstream from the bidirectional origin is a scaffold-attached fragment of 3.4 kb [35]. It may be that there are multiple initiation sites in the 28 kb domain, which as a whole may represent a single complex origin; if so, the scaffold-attachment site could well be an integral component of a broadly dehned initiation region [36**]. Replication forks, as detected by nascent, pulse-labelled DNA, are associated with the nuclear matrix scatfold prepared either by extraction with high salt concentrations or with lithium 3’,5’diiodosalicylate [37*], providing a tool for isolating replicative intermediates for mapping origins by two-dimensional gel electrophoresis. The as sociation of replicating DNA is transient and should be differentiated from the sequence-dependent interactions seen with SARs.The localization of replication forks with a nuclear substructure probably reflects the foci of replication forks detected by probes specific for newly replicated DNA, detected in reconstituted Xenopus sperm nuclei in vitro [38] and in fractionated kangaroo or mouse cells [39]. Roughly 300 foci were dispersed throughout the nucleus, implying that each fluorescent spot could reflect several hundred replication complexes. Recent work shows that nuclear formation is a general prerequisite for initiation of DNA replication in the Xenopus extract system [40-l, and that the foci initiate coordinately within a membrane-defined nucleus or group of nuclei [41*]. While the nuclear membrane appears to play a role in
Fig. 3. Initiation and bidirectional replication forks bound to a nuclear substructure. A representation of progression through initiation of DNA replication and bidirectional elongation, where both the elongation complex and the origin remain associated with a nuclear substructure tin this case plane PI The shaded oval is the origin of replication, the regions marked T are the termination sites, and the white ovals are the replication forks. The newly synthesized strand is dotted. Not all the DNA is shown in the central third for the sake of simplicity. The progression of time is indicated by the arrow tl to t2. Adapted from 1421.
411
412
Nucleus
and gene expression
del%ng a replicating unit, the foci of DNA syfithesis are not membrane associated. There is no information on the biochemical nature of the structure that this organization reflects, yet it is an elegant visual con&nation that nuclear organization is involved in the coordination of replication events. The nuclear framework for DNA replication must as yet be represented as a simple plane (Fig. 31, yet it will become increasingly more detailed as probes for the replication machinery become available.
10.
SCHMIDT AM4 HE-I-ERICH SU, KRAUSS G: A Single-stranded DNA-binding Protein from S cereulslae Specilically Recog nizes the T-rich Strand of the Core Sequences of AR.5 Elemerits and Discriminates Against Mutant Sequences. EMBO J 1991, lO:981-985. Identification of a 65 kD single-strand DNA.bindlng protein that binds the ARS consensus sequence. Point mutations in the consensus lower binding alfinity. Poly U and poly dT sequences also bind with lower affinities than the ARS consensus. No double.strand DNA binding is observed
..
11. ..
WAU(ER SS. FRANCE~CONI SC, EISENRERG S: A DNA Replication Enhancer in Saccbaromyces cerevisfae. Proc Null Acud Sci
USA 1990, 87:4665-&69.
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The effects of OBFI and of near matches to the AR.5 con.sensus are analysed by linker mutagenesis at the telomeric ARS 121. OBFl -binding sites function over a distance in either orientation as a replication-enhancer element. Near matches can be ablated without total loss of AR.5 function.
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HOLVE~ SG. S~IIIH MM: Interaction of the H4 Autonomously Replicating Sequence Core Consensus Sequence and its 3’Flanking Domain. Mel Cell Biol 1989, 95464-5472. One of the most complete mutagenesis studies on the flanking se quences required for ARS function. Elimination of all near matches to the ARS consensus greater than 8111 still permits plasmid replication, although inversion of the 11’ 11 consensus does not.
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VAN HOLTEN JV. NEWIDN CS: Mutational Analysis of the Consensus Sequence of a Replication Origin from Yeast Chromosome Ill. Mol Cell Biol 1990, 10:3917-3925. The most complete mutagenesis study in existence on the yeast ARS consensus. Changes at nearly every nucleotide modify or eliminate replication efficiency. Flanking residues influence ARS efficiency and the context of the consensus also alters the effects of mutations in the consensus itself. l .
KLPUNG D, KF!XCXY SE: Reversion of Autonomously Replicating Sequence Mutations in Saccbaromyces cereuisiae Creation of a Eucaryotic Replication Origin within Procaryotic Vector DNA Mol Cell Biol 1990, 10:26%272. A surprising genetic result showing that reversion of ARSdeficient plas mids to replication-competent ones, involves the creation of near. matches to the consensus in vector DNA Strong arguments are made for the role of the ARS consensus.
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I WANG 1X. KORNIXRG A A Novel Protein Binds a Key Origin Sequence to Block Replication of an .E. coli Minichromosome. Cell 1990, 63:325-331. A protein hzs been puntied from E. coli that hinds speciiically to the consened 13.mcr motif. It appears to be L negative regulator of the Initiation of DNA replication that functions by prt?rnting the opening of the helix at this site. 17.
JFK, GMSER SM: Identification and Purification of .. a Protein that Binds the Yeast ARS Consensus Sequence. cell 1991. 64:951-&o. A protein that recognizes the yeast AR5 consensus is purified and binding sites are characterized. The protein (ACBP) binds preferentially to the T-rich single strand of the consensus. Point mutations in the AR.5 consensus ate mapped which affect affinity and binding sites on genomic ARS elements.
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10:5442-5454. AR.5 elements of S. pombe are shown to bind to S. pornbe nuclear scaf folds. Dmrophifa SARs bind scaffolds from both yeast species, and sev etal Drosophila SARs support plasmid replication in both. Common sequences appear to be required for both replication and scaffold binding.
Replication 21.
SURDEJ P, GOT C, ROSSET R, MIASWD R: SupragenIc Loop ChganIzatlon: Mapping, in Drosophila Embryos of ScatfoldAssociated Regions on a 800 Kilobase DNA Continuum Cloned from the 14B-I5B First Chromosome Region. Nucleic Acids Res 1990, 18:371+3722.
22. .
BRUN C, DANC Q, MM~SOD R: Studies on a 800kb DNA Stretch of the Drosophila X Chromosome: Comapping of a Subclass of Scaffold Attached Regions with Sequences able to Replicate Autonomously in Yeast. Ato1 Cell Biol 1990, 10:54555463. Drosophila SAPs that were identified in a 800 kb walk on chromosome X, are analyscd for their ability to promote autonomous replication in yeast. The results lend statistical support to the argument presented in 12091. PICK 1 IA~OCHE T, GAYER SM: Nuclear Scaffold Stimulates, but is not Essential for ARS Activity in Succhammyces cereuisim Analysis of the Drosophila ftz SAR. EMUOJ 19’90, 9:40074016. The relationship between scaifold attachment and ARS activity is explored with a series of deletions and artificial constructs with the & SAR. ARS activity is stimulated by the presence of sequences that bind the scaffold. Oligo-dT stretches are characteristic of both activities.
23.
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Ah&in B. Attachment
KA? E. I~ALRALDE E, h!SMhtU Binding to Nuclear Scaifolds
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ADACIII Y. KAS E. IAEMMU UK: Preferential Cooperative Binding of DNA Topoisomerase II to Scaffold-Associated Regions. E>lBO J 1989, 8:39974006. Topoisomerase II is shown to bind coopemtively to Drosopbih SAR fragments. This interaction reflects a preference of topoisomemse II for the narrow minor-groove structure of DNA containing oligo dA or dT stretches as tidenced by preferred binding to dA, ,T, , polymers and inhibition of the interaction by disramycin. .
26.
MIEIJW C, KOHV~ Y. K~HVUI.~HI(;I;MA~~~I T, Bong J: Hierarchical Binding of DNA Fragments Derived from ScaifoldAttached Regions: Correlation of Properties In Vitro and Function In Vito. Hiochemisf~ 1990, 2917475-7485. A repeated sequence motif (ATATITI of the human interferon h 5’ SAR was tested for binding to scaffolds in a multimeric state. Mutation of the motif reduces but does not eliminate interaction. .
27.
VON KRES JP. PHI-VAN L DIEKMANN S. SIRATLING WH: Curved Chicken Lysozyme 5’ Matrix Attachment 3’ Followed by a Strongly Curved DNA Sequence. Acids Res 1990, 18:388-3885.
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HECK MMS, SPRALWNC AC: Multiple Replication Origins are Used During Drosophila Chorion Gene AmpliIication. J Cell t3iol 1390, 110:90>914. The first published report of mapping of a higher eukaryotic origin by two-dimensional gel electrophoresis techniques. More than one origin is found to fire within a 7.7 kb region in the Dms@ih chorion gene cluster.
30.
..
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DAILF( L, CADDE MS, HEINI-Z N, HEINIZ NH: PurBication of RIP60 and RIPlOO, Mammalian Proteins with Origin-Specific DNA-Binding and ATP-dependent DNA Helicase Activities. Mol Cell Biol 1990, 10:622%235.
and attachment
sites Casser
repeats downstream of the bidiwas shown to be 60 kD ln size. It of roughly 100 kD.
CADDE MS, DAILEY L, H!mm NH: RI&O. a MammaEan Origin-Binding Protein, Enhances DNA Bending Neat the Dihydrofolate Reductase Origin of Replication. Mel Cell Brd 1990, 10:623&6243. The 6OkD protein that binds near or in the DHFR origin region was shown to enhance the bend of stably bent DNA near its binding site.
32.
.
BURHANS WC, VASSILR, LT, CADDE MS, HEIPSIZ NH, DEPAMPH~~~ ML Identification of an origin of Bidirectional DNA Rep& cation in Mammalian Chromosomes. Cell 1990. 62:955+65. An origin of bidirectional replication was mapped to roughly 450 bp by a new method that can capitalize on the fact that Okazald fragments are only synthesized on the lagging strand. This is the most closely mapped mammalian origin to date.
33. ..
34.
FARACHE G, RABIN SV, R~E~~~~~KI-W~INY J. MORFAU J, TARGA FR, SCHERRER K: Mapping of Structural and TranscriptionRelated Matrix Attachment Sites in the Alpha-GlobI Gene Domain of Avian Etythroblasts and Etythrocytes. Mol Cell Bid 1990,10:53495358.
35.
DIJ~?VEL PA, HAMUN JL Matrix Attachment Regions are Positioned Near Replication lnitiation Sites, Genes and an Interamplicon Junction in the AmpIiIied Dihydrofolate Reductase Domain of CHO Cells. Mel Cell Bid 1988, 8:5398-5409.
210:587-599.
25.
factors
A protein that recognizes the (ATT), rectional origin in the DHFR domain cofractionates with a helicase activity
Inhibition of DNA HI by Distamycin.
Artificial AT-rich constructs and Drmophilu SARs are examined for scaf fold binding and interaction with histone Ill. Rigid DNA with narrow minor-groove structure (e.g. dA, ,T, ,) has higher affinity than altemating dAT. Both SAPS and artificial constructs can he competed by distamycin.
orinins,
VAUGHN JP, DIJhw’rzL PA, HAMIJN JL Replication Initiates In a Broad Zone in the Amplitied CHO Dihydrofolate Reductase Domain. Cell 1990. 6I:IO75Io87. The two-dimensional gel electrophoresis system was used to map replication origins in the amplified DHFR domain. A broad region covering nearly 28 kh contains structures that typify the origin bubble, suggesting that DNA replication initiates here at many sites.
36.
..
VALICHN JP, DIJKWEL PA MUUENIIERS IHF, HM~LLN JL Replication Forks are Associated with the Nuclear Mattix. Nucleic Acids Res Im. 18;1365-1969. Newly synthesized DNA was shown by two-dimensional gel electrophoresis to be enriched in the nuclear matrix fraction prepared by various extraction procedures. This rules out the criticism of high-salt artefacts in earlier studies and provides a way to enrich for repllcative intermediates in mammalian nuclei for twodimensional gel analysis. 37. .
38.
MIUS AD, BLOW JJ, WHITE JG, AMOS WB. WILCOCK D, LWCEY RA: Replication Occurs at Discrete Foci Spaced Throughout Nuclei Replicating In Vitro. J Cell Sci 1989, %:471-477.
39.
NAKAYASU H, BERFZNEY R: Mapping Replicational Sites Eucatyotic CelI Nucleus. J Cell Biol 1989, 108:1-11.
40. .
Brow
JJ. SLEEMAN AM: Replication
pus Egg Extract is Dependent Sci 1990, 95:383391.
In a Xenopus extract/nuclear reconstituted nuclei perform branes and the use of mitotic and DNA replication in vitro
in the
of Purified DNA in Xeno on Nuclear Assembly. J Cell
reconstitution assay it is shown that only DNA replication. The depletion of memextracts eliminate nuclear reconstitution
41. .
&NO GH, the Timing
&K!zY RA: The Nuclear Membrane Determines of DNA Replication in Xenopus Egg Extracts. J Cell Biol 1991, 112:557-566. Chick erythrocyte nuclei form ‘multinuclear aggregates’ in cell-free Xenopus extracts. Such nuclei lack their individual nuclear membranes but share a perimeter envelope. These nuclei undergo DNA replication synchronously at foci distributed throughout the aggregate. 42.
DINGMAN CW Bidirectional Topological Considerations.
SM Gasser, Swiss Institute 1066 Epalinges s/Iausanne,
Chromosome
J 7kor
for Experimental Switzerland.
Replication: Some 43:187-195.
Biol 1974,
Cancer
Research
(ISREC),
413
origins,
factors and attachment
sites
Susan M. Gasser Swiss Institute
for Experimental
Cancer
Research,
Lausanne,
Switzerland
The initiation of eukaryotic DNA synthesis occurs at specific sites determined by both cis- and tram-acting elements. Here I review advances in the characterization of yeast origins, origin-binding proteins and the relationship of DNA replication to nuclear substructure in yeast.
Current
Opinion
in Cell
Introduction Our ability to reconstitute the cellular machinery for DNA replication in z&-o has progressed enormously in the past few years. Replication of simian virus 40 DNA can be reconstituted completely in a soluble reaction containing purified factors of the cellular replication machinery and the virally encoded initiator protein, large T antigen [I**]. Much of what we have learned, including the regulation of the system through phosphorylation and dephosphoryiation events, is directly applicable to genomic replication. However, the most important control point in genomic DNA synthesis - its initiation -will remain unexplained until the genomic sites of initiation and the proteins that recognize them are fully characterized. In the following pages I present recent advances in our understanding of the sequences required for initiation of DNA replication at yeast autonomously replicating sequence (ARS) elements, the proteins that bind origins, and some ultrastructural aspects of replication origins.
The yeast and ARS consensus AR.5elements were first isolated from yeast genomic DNA and from the endogenous 2micron circle because of their ability to increase the transformation efficiency of yeast integrative plasmids (reviewed in [ 2,3]). ARS elements allow the autonomous replication of large numbers of plasmid.s, with an unstable mitotic phenotype. The replication of ARS plasmids, like that of genomic DNA, requires entry into S-phase and initiation occurs only once per cell cycle. Proof that initiation occurs at or near ARS elements came from a two-dimensional gel electrophoresis system that allows separation of replication intermediates generated by the restriction digestion of replicating plasmids [4]. With this method, and a complementary neutral/alkaline system [ 51. ARSelements and genomic origins have been mapped on the ring-deriva-
Biology
1991,
3:407413
tive of chromosome III of Sacdaromyces cerevbiue. Five out of ten mapped ARS elements are used as chromosomal origins, and four others remain to be tested [2]. There are, however, no origins without ARS activity, and at least one ARS is necessary for maintenance of the circular chromosome III. All ARS elements contain one or more copies of a perfect or near-perfect match to an 11 bp A/T-rich sequence (WTITATRTTTW[W=AorT;R=AorG])thatisnecessary but not sufficient for ARS activity. In addition, A/Trich flanking DNA is required. The fact that single basepair mutations could abolish replicative activity suggested the importance of the ARS consensus [6]. However, single mismatches appear to be tolerated at many other ARS elements. To resolve this apparent contradiction, Van Houten and Newlon [7**] have mutated, to each of the three other possibilities, every nucleotide in the ARS consensus of a semisynthetic ARS element derived from the C2Gl ARS (now designated ARS307). These 33 point mutations were reintroduced into centromere-contaming vectors carrying the SUP11 and URA3 markers to allow quantitation of high-frequency transformation and plasmid-loss rate under non-selective conditions. Every nucleotide was found to be important for maximal ARS function, including, but to a lesser degree, the residue in position 12 and three bases located 5’ of the consensus (Fig. 1). Despite this strong demonstration of the significance of each nucleotide within the C2Gl ARS consensus, comparison with natural genomic ARS elements shows that the requirements are at least partially dependent on context. Among all sequenced ARS elements, only positions 7, 8, and 11 appear invariant. Thus, in certain contexts some, but not all, point mutations can be tolerated. Kipling and Kearsey [8**] have obtained results that underscore the importance of the ARS consensus using random mutagenesis to recover a replicating plasmid from non-replicating derivatives of an Ml3 vector. In two regions of the non-replicating Ml3 vector (not the region
Abbreviations ACBP-ARS
consensus-binding
protein;
ARS-autonomously SAR-scaffold
@ Current
Biology
replicating attached region.
sequence;
Ltd ISSN 0955*74
DHFR-dihydrofolate
reductase;
407
408
Nucleus
and gene expression
.
The ARS consensus-binding
protein
(a)
1
2
3
4
5
6
7
8
9
10
A
T
T
T
7
8
9
10
11
6) T 1
2
3
A 4
5
6
11
(c)
T
c
T
1
2
3
4
5
6
7
8
9
10
A/r
T
T
T
A
T
A/G
T
T
T
1
2
3
4
5
6
7
8
9
10
Two groups have identified a nuclear activity from S cerezvkiue that wiII recognize an oligonucleotide con taining the yeast ARS consensus [9**,10**]. In one cast the hydroxy radical footprint was determined on the H4 ARS element, the protein was purified, and the binding specificity of the 67 k~ protein for several genomic m elements and mutant ARS consenses was characterized [9-l. Although genetic evidence is lacking, the following observations are highly suggestive of a role in the initiation of DNA .synthesis.
11
(d) A/r 11
Fig. 1. A summary of important bases in the yeast autonomously replicating sequence (ARS) consensus sequence. Mutagenesis studies (a), reversion studies (b). and ARS consensus-binding protein (ACBP) studies (c) have contributed to the evaluation of the relative importance of the different residues in the yeast ARS consensus given here. The wild-type consensus sequence is indicated in (d) and the position of each residue is numbered. (a) For the C2Cl ARS, mutations at the following six positions within the core consensus resulted in an inactive ARS: from thymidlne (T) at positions 3, 8, 9 and 10; from adenosine (A) at position 5; and from guanine U to a pyrimidine at position 7. Mutations of the thymidines at positions 2 and 4 to adenosines weakened but did not eliminate ARS activity. Mutation to a base not inherent in the consenses sequence appeared to be tolerated in one instance only: thymidine to cytosine (C) at position 6. All other variation produced functional mutations (0). Cl, partially active mutation; n , inactive mutation. (b) Reversion studies in the Ml3 vector show that, for functional ARS consensus, six out of 11 bases are invariably wild type. These are identical to the invariant bases described in (a) and are given here in bold type. (c) Interaction of ACBP with. the wild-type consensus is greatly reduced by point mutations (given in bold type) at positions 3, 7 and 10. There is one permitted mutation (encircled) at position 5 where thymidine replaces the consensus adenosine. Intriguingly, this replacement is the only alternative base found at position 5 in naturally occurring ARS consenses: ARS121, ARS305, and ARS309 all contain thymidine at position 5.
containing a mutant yeast ARS element) the point mutations that confer AR.5activity were found to improve 9/l 1 near matches to the ARS consensus to lo/11 matches. Each mutation occurred in a region containing multiple 9/l 1 matches. The conserved positions among these ‘spontaneous’ ARS consenses are compared with the results of Van Houten and NewIon in Fig. 1. Remarkably, many of the conserved nucleotides common to these nvo studies are also conserved in the binding sites mapped for the AR.5 consensus-binding protein (ACBP) isolated by Hofmann and myself (9*=]. The consenlation of the AR.5 consensus argues for a consensus-binding protein, which would in some sense serve as the cellular analogue of T antigen or the bacterial DnaA initiation protein.
Footprint analysis irz llitro showed asymmetric protection of the 11 bp ARS consensus and adjacent sequences at the H4 ARS. A protein in the yeast scaffold extract bound specifically to the double-stranded form of a 22 bp oIigonucleotide containing the consensus and binds with higher af%nity to the T-rich, but not the A-rich, single strand of the same sequence. An excess of EscberidJiu coli single-strand DNA did not compete for the binding, whereas it competed eficiently for a major non-specific single-strand binding activity in the nuclear extract. The factor wa purified to apparent homogeneity as a 67kD polypeptide, ADBP, that retains the described DNA-binding properties, even after renaturation from a sodium dodecylsulphate gel. Apart from the oIigonucleotide, ACBP binds singlestranded fragments containing the perfect ARS consensus from four genomic ARS elements. Sequence specificity could be shown by quantifying the ability of the wild-type ARS consensus to compete for the binding of ollgonucleotides containing point mutations in the 11 bp ARS consensus within the semisynthetic C2Gl AR.5 element from the study by Campbell and Newlon [ 21. Three of the four point mutations that were tested significant11 reduced the affinity of the AR!%binding factor for the ARS consensus, while one (an A-T mismatch) appeared to enhance the affinity of ACBP for the consensus (Fig. 1). In addition, the affinity of ACBP for the ARS consensus appears to be stimulated by the presence of either of the leading-strand polymerases from yeast (6 or E) but not by DNA polymerase a or topoisomerase II. Unlike T antigen, ACBP has no detectable ATP-binding or helicase activity in its purified state (JFX Hofmann and SM Gasser, unpublished data). The significant variation of binding affinities due to single nucleotide changes demonstrates that the ACBP-DNA interaction is sequence specific. Within the range of its specificity, however, are certain 9/11 matches to the ARS consensus in single-stranded form. Near-match binding sites have been mapped in the 3’.flanking domains of the genomic ARSl and H4 ARS elements, and in each case the 9/11 binding site is on the opposite strand 3’ of the perfect consensus. At the H4 ARS, the two ACBP binding sites correlate with the boundaries of the minimal-functional AR.5element determined by deletion analysis [ 121. A model is presented in Fig. 2 that suggests a role for
Replication
the 3’ orientation of binding sites in the initiation of DNA synthesis.
(a) Recognition
and targeting
m (b) Helicase-mediated
unwinding
tc) Targeting polymerases leading-strand
of the 3’ element
for bidirectional synthesis
Fig. 2. Putatrve roles for an ARS consensus binding protein (ACBP) and the 3’ flank in the initiation of DNA replication. It is proposed that the autonomously replicating sequence (AR9 binding factor (represented by circles) first recognizes the double-strand ARS consensus and targets an associated helicase to the origin (a). Local unwinding in the 3’ DNA unwinding element (DUE) 1131 is Indicated in (b). ACBP should then recognize the T-rich strand of the perfect consensus and of the 3’ near-match to the consensus. It IS generally thought tin analogy to bacterial and viral systems) that synthesis starts through extension of an RNA primer by the DNA polymeraseu-primase complex. A transition to the leading strand polymerase ll**l, might then take place at the ARS consensus and its 3’ near match, through interaction of the leading strand polymerases 6 or E (~016, polo) with ACBP (c). The leading strand synthesis is indicated by the black arrows pointing in a 5’ to 3’ direction. Bidirectional replication may require two ACBP binding sites with 3’ orientation to each other.
origins,
The 3’ flanking
factors
and attachment
sites Gasser
domain
The observations on ACBP binding sites, and extensive deletion and mutagenesis studies, contribute to the two models put forth to explain the role of the DNA 3’ of the T-rich ARS consensus, a region that complements the ARSconsensus to provide replicative activity on plasmids. It has been shown that the 3’ region is hypersensitive to single-strand-specific endonucleases in negatively supercoiled plasmid DNA [3,13]. It would thus be a region of preferred unwinding in the presence of a helicase, and could serve as an entry site for the replication machinery. Umek and Kowalski [14*] have shown in viva that thermal energy (growth at 30°C as opposed to 23°C) can suppress mutations in the 3’ flanking region that decrease its propensity to unwind. This confirms that the double helix must unwind for origin hmction in zklo. The study of unwinding elements has been extended to the E. coli oriC, where three tandem repeats of a highly conserved 13-mer were suggested to serve as protein-binding sites and as the initial site of helix unwinding. The leftmost 13.mer can be replaced with another helically unstable sequence that contains only four matches to the consensus. This suggests that the potential for unwinding is more important than its sequence for one of the 13.mer repeats [15-l. On the other hand, a 13.mer-specific DNA-binding protein has recently been purified [ 16**]. This suggests that the repeated 13-mers serve both as protein-binding sites and as an easily unwound domain. A similar explanation is likely to be true for the yeast ARS 3’ flanking region. It has been suggested [ 171, that the essential contribution of the 3’ flank was multiple nearmatches to the ARS consensus, usually in an inverted onentation, which would provide binding sites for the then putative ARS-binding factor. The reversion studies on the Ml3 plasmid appear to support this idea, as mutagenesis created AR.5elements where multiple near matches were available on the opposite strand [ES**].Moreover, the nine genomic ARS elements presented by Palzkill and Newlon [ 171 and the telomeric ARS 121, studied by Walker et al. [ 1I**], all contain a 3’.flanking region with 9/l 1 matches to the ARS consensus inverted with respect to the T-rich strand of the major consensus site. In addition to preserving inverted ARS consenses, all these regions show sensitivity to single-strand-specific nucleases, indicative of an easily unwound domain. In some cases, linker mutagenesis and multiple point mutations in these near-perfect consenses seem to question the importance of the 3’ region as a protein-recognition site, at least in plasmid replication. At the telomeric ARS, mutagenesis of all three near matches decreases AR.5efhciency IO-fold but does not eliminate it [ 11 l *I At the H4 ARS, T-A mutations that maintain the propensity of the region to unwind, but at the same time destroy the nearmatch consenses, are still functional for plasmid replication [ 120*]. There are two possible explanations that remain consistent with a dual role for the 3’ flank: either the downstream binding sites are necessary only for the initiation of bidirectional DNA synthesis, such as one finds in
409
410
Nucleus
and gene expression
the chromosome (Fig. 21, while unidirection’al synthesis is sufficient for plasmid maintenance, or the run of seven Ts (or other 250 bp) of ‘SAR-DNA’ for efficient and detectable interaction with scaffolds, Within the ftz SAR, the ability to bind the scatTold in vitro correlated with enhanced plasmid replication, but was not a strict prerequisite for it 123’1.
Scaffold attachment may stimulate ARS activity in several ways. The sctiold-binding sequence may localize the ongin to a subcompartment of the nucleus that is rich in the factors and enzymes necessary for the initiation of DNA synthesis. In view of the AT-rich character of the SAR DNA, it may also coincide with the unwinding element discussed above, facilitating the opening of the double helix at the origin for entry of primase and polymerase activities, although scaffold attachment itself is not dependent on unwound or single-stranded DNA (T Falquet, personal communication). Finally, the SAR may provide repeated binding sites for accumulation of a specific initiation factor, such as ACBP, which itself fractionates with the scaffold fraction. Kiis et al. [24*] have also examined the scaffold binding capacity of AT-rich artificial polymers, and suggested that the oligo dT sequences and narrow minor-groove structure is the critical factor for scaffold interaction. They showed that distamycin, a drug that interacts specifically with a narrow minor-groove structure, competes efficiently for &AR-scaffold interaction. Moreover, the checkered dA,,Til polymer, which forms a rigid helix with a typically narrow minor groove, showed high affinity for the scaffold, provided it was sufficiently long ( > 160 bp). The same structural preference could be observed in the cooperative interaction of topoisomerase Il with SARs and artificial sequences in vitro [ 25.1. Studies on human and chicken &AR-scaffold interactions have come to similar conclusions [26*,27].
In summary, SARs and ARS elements share multiple structural properties. Eckdahl and Anderson [28] have pointed out that dA or dT stretches of threesix nucleotides in length, occurring at 10-11 bp intervals (resulting in bent DNA) and at 6-8 bp intervals (resulting in a rigid structure), are statistically enriched in yeast ARS elements and prokaryotic origins. These are identical with the sequences that characterize scaffold binding sites. Two important questions remain: whether the correlation between origins and scaffold attachment holds in
Replication
higher eukaryotes, and whether the SARs actually define the bases of DNA loops in intact cells.
A glance
at higher
eukaryote
origins
Sequence analysis of dihydrofolate reductase (DHPR) origin region reveals the presence of several long stretches of poly dT or dA sequence, stretches of alternating purine/pyrimidine pattern, and regions with distinctly bent character [29]. Several non-B-form DNA structures are favoured by the region, notably formation of triple helices and an unwound double helix. Some of the motifs found in the DHPR origin are also found in the two replication origins mapped by two-dimensional gel electrophoresis in the Drosophifiu chorion locus [30**] - in particular, the repetition of oligo dT stretches forming a region of bent DNA and near matches to the yeast ARS consensus. Intriguingly, a protein called RIPGO,purified from HeIa cell extracts, binds specifically to a repeated sequence element (ATT),, that occurs twice near the origin of bidirectional replication at DHPR, as well as recognizing the yeast ARS consensus sequence in z&-o [31-l (N Heintz, personal communication). It is not known whether these motifs are important for DNA replication at this locus, but the fact that RIPGOcopurihes with an ATP-dependent helicase activity is highly suggestive of a role in an initiation complex [31**]. The RIP60 protein itself enhances bending at its binding site, which is adjacent to a region of naturally bent DNA, a feature that often coincides with scaffold binding activity [24*,32*,33**]. Besides sequence homologies, there is experimental evidence that the origins of replication located 5’ of the
origins,
factors
and attachment
sites Casser
chick u-globin gene and 3’ of the Chinese hamster DHPR gene are both close to scaffold-attachment sites [34,35]. At least two origins of replication have been mapped within a 28 kb region downstream of the DHFR gene (see Hamlin et al., this issue pp 414-423), and the more gene-proximal initiation site appears to promote bidirectional replication [33**]. Several kilobases downstream from the bidirectional origin is a scaffold-attached fragment of 3.4 kb [35]. It may be that there are multiple initiation sites in the 28 kb domain, which as a whole may represent a single complex origin; if so, the scaffold-attachment site could well be an integral component of a broadly dehned initiation region [36**]. Replication forks, as detected by nascent, pulse-labelled DNA, are associated with the nuclear matrix scatfold prepared either by extraction with high salt concentrations or with lithium 3’,5’diiodosalicylate [37*], providing a tool for isolating replicative intermediates for mapping origins by two-dimensional gel electrophoresis. The as sociation of replicating DNA is transient and should be differentiated from the sequence-dependent interactions seen with SARs.The localization of replication forks with a nuclear substructure probably reflects the foci of replication forks detected by probes specific for newly replicated DNA, detected in reconstituted Xenopus sperm nuclei in vitro [38] and in fractionated kangaroo or mouse cells [39]. Roughly 300 foci were dispersed throughout the nucleus, implying that each fluorescent spot could reflect several hundred replication complexes. Recent work shows that nuclear formation is a general prerequisite for initiation of DNA replication in the Xenopus extract system [40-l, and that the foci initiate coordinately within a membrane-defined nucleus or group of nuclei [41*]. While the nuclear membrane appears to play a role in
Fig. 3. Initiation and bidirectional replication forks bound to a nuclear substructure. A representation of progression through initiation of DNA replication and bidirectional elongation, where both the elongation complex and the origin remain associated with a nuclear substructure tin this case plane PI The shaded oval is the origin of replication, the regions marked T are the termination sites, and the white ovals are the replication forks. The newly synthesized strand is dotted. Not all the DNA is shown in the central third for the sake of simplicity. The progression of time is indicated by the arrow tl to t2. Adapted from 1421.
411
412
Nucleus
and gene expression
del%ng a replicating unit, the foci of DNA syfithesis are not membrane associated. There is no information on the biochemical nature of the structure that this organization reflects, yet it is an elegant visual con&nation that nuclear organization is involved in the coordination of replication events. The nuclear framework for DNA replication must as yet be represented as a simple plane (Fig. 31, yet it will become increasingly more detailed as probes for the replication machinery become available.
10.
SCHMIDT AM4 HE-I-ERICH SU, KRAUSS G: A Single-stranded DNA-binding Protein from S cereulslae Specilically Recog nizes the T-rich Strand of the Core Sequences of AR.5 Elemerits and Discriminates Against Mutant Sequences. EMBO J 1991, lO:981-985. Identification of a 65 kD single-strand DNA.bindlng protein that binds the ARS consensus sequence. Point mutations in the consensus lower binding alfinity. Poly U and poly dT sequences also bind with lower affinities than the ARS consensus. No double.strand DNA binding is observed
..
11. ..
WAU(ER SS. FRANCE~CONI SC, EISENRERG S: A DNA Replication Enhancer in Saccbaromyces cerevisfae. Proc Null Acud Sci
USA 1990, 87:4665-&69.
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The effects of OBFI and of near matches to the AR.5 con.sensus are analysed by linker mutagenesis at the telomeric ARS 121. OBFl -binding sites function over a distance in either orientation as a replication-enhancer element. Near matches can be ablated without total loss of AR.5 function.
reading
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HOLVE~ SG. S~IIIH MM: Interaction of the H4 Autonomously Replicating Sequence Core Consensus Sequence and its 3’Flanking Domain. Mel Cell Biol 1989, 95464-5472. One of the most complete mutagenesis studies on the flanking se quences required for ARS function. Elimination of all near matches to the ARS consensus greater than 8111 still permits plasmid replication, although inversion of the 11’ 11 consensus does not.
T, MELENDY T, S~UMAN B: Sequential Initiation of Lagging and Leading Strand Synthesis by Two Different Polymerase Complexes at the SV40 DNA Replication
TSUIUMOTO
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A complete reconstitution of simian vints 40 replication with only pun lied components. DNA polymetases a and 6 are shown to be required for synthesis; a polymerase switching model is proposed.
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VAN HOLTEN JV. NEWIDN CS: Mutational Analysis of the Consensus Sequence of a Replication Origin from Yeast Chromosome Ill. Mol Cell Biol 1990, 10:3917-3925. The most complete mutagenesis study in existence on the yeast ARS consensus. Changes at nearly every nucleotide modify or eliminate replication efficiency. Flanking residues influence ARS efficiency and the context of the consensus also alters the effects of mutations in the consensus itself. l .
KLPUNG D, KF!XCXY SE: Reversion of Autonomously Replicating Sequence Mutations in Saccbaromyces cereuisiae Creation of a Eucaryotic Replication Origin within Procaryotic Vector DNA Mol Cell Biol 1990, 10:26%272. A surprising genetic result showing that reversion of ARSdeficient plas mids to replication-competent ones, involves the creation of near. matches to the consensus in vector DNA Strong arguments are made for the role of the ARS consensus.
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JFK, GMSER SM: Identification and Purification of .. a Protein that Binds the Yeast ARS Consensus Sequence. cell 1991. 64:951-&o. A protein that recognizes the yeast AR5 consensus is purified and binding sites are characterized. The protein (ACBP) binds preferentially to the T-rich single strand of the consensus. Point mutations in the AR.5 consensus ate mapped which affect affinity and binding sites on genomic ARS elements.
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8:210-225.
AMA~
B. GAWR SM: Drosophila Scaffold Attached Regions Bind Nuclear Scaffolds and Can Function as ARS Elements in Both Budding and Fission Yeasts. Mol Cell Biol 19W,
10:5442-5454. AR.5 elements of S. pombe are shown to bind to S. pornbe nuclear scaf folds. Dmrophifa SARs bind scaffolds from both yeast species, and sev etal Drosophila SARs support plasmid replication in both. Common sequences appear to be required for both replication and scaffold binding.
Replication 21.
SURDEJ P, GOT C, ROSSET R, MIASWD R: SupragenIc Loop ChganIzatlon: Mapping, in Drosophila Embryos of ScatfoldAssociated Regions on a 800 Kilobase DNA Continuum Cloned from the 14B-I5B First Chromosome Region. Nucleic Acids Res 1990, 18:371+3722.
22. .
BRUN C, DANC Q, MM~SOD R: Studies on a 800kb DNA Stretch of the Drosophila X Chromosome: Comapping of a Subclass of Scaffold Attached Regions with Sequences able to Replicate Autonomously in Yeast. Ato1 Cell Biol 1990, 10:54555463. Drosophila SAPs that were identified in a 800 kb walk on chromosome X, are analyscd for their ability to promote autonomous replication in yeast. The results lend statistical support to the argument presented in 12091. PICK 1 IA~OCHE T, GAYER SM: Nuclear Scaffold Stimulates, but is not Essential for ARS Activity in Succhammyces cereuisim Analysis of the Drosophila ftz SAR. EMUOJ 19’90, 9:40074016. The relationship between scaifold attachment and ARS activity is explored with a series of deletions and artificial constructs with the & SAR. ARS activity is stimulated by the presence of sequences that bind the scaffold. Oligo-dT stretches are characteristic of both activities.
23.
.
24. .
Ah&in B. Attachment
KA? E. I~ALRALDE E, h!SMhtU Binding to Nuclear Scaifolds
J Mel Biol
1989
UK: Specitic and Histone
ADACIII Y. KAS E. IAEMMU UK: Preferential Cooperative Binding of DNA Topoisomerase II to Scaffold-Associated Regions. E>lBO J 1989, 8:39974006. Topoisomerase II is shown to bind coopemtively to Drosopbih SAR fragments. This interaction reflects a preference of topoisomemse II for the narrow minor-groove structure of DNA containing oligo dA or dT stretches as tidenced by preferred binding to dA, ,T, , polymers and inhibition of the interaction by disramycin. .
26.
MIEIJW C, KOHV~ Y. K~HVUI.~HI(;I;MA~~~I T, Bong J: Hierarchical Binding of DNA Fragments Derived from ScaifoldAttached Regions: Correlation of Properties In Vitro and Function In Vito. Hiochemisf~ 1990, 2917475-7485. A repeated sequence motif (ATATITI of the human interferon h 5’ SAR was tested for binding to scaffolds in a multimeric state. Mutation of the motif reduces but does not eliminate interaction. .
27.
VON KRES JP. PHI-VAN L DIEKMANN S. SIRATLING WH: Curved Chicken Lysozyme 5’ Matrix Attachment 3’ Followed by a Strongly Curved DNA Sequence. Acids Res 1990, 18:388-3885.
28.
ECKDAHI. IT, ANDEKX)N JN: Conserved DNA Origins of Replication. Nucleic Acids Res 1990,
29.
CADDLE Triplexes. Initiation Replicon.
A NonSite is Nucleic
Structures 18:16091612.
In
MS, LUWER RH, HEU‘SI% NH: intramolecular DNA Bent DNA and DNA Unwinding Elements ln the Region of an AmpliEed Dihydrofolate Reductasc J Mel Biol 1990, 211:19-33.
HECK MMS, SPRALWNC AC: Multiple Replication Origins are Used During Drosophila Chorion Gene AmpliIication. J Cell t3iol 1390, 110:90>914. The first published report of mapping of a higher eukaryotic origin by two-dimensional gel electrophoresis techniques. More than one origin is found to fire within a 7.7 kb region in the Dms@ih chorion gene cluster.
30.
..
31. ..
DAILF( L, CADDE MS, HEINI-Z N, HEINIZ NH: PurBication of RIP60 and RIPlOO, Mammalian Proteins with Origin-Specific DNA-Binding and ATP-dependent DNA Helicase Activities. Mol Cell Biol 1990, 10:622%235.
and attachment
sites Casser
repeats downstream of the bidiwas shown to be 60 kD ln size. It of roughly 100 kD.
CADDE MS, DAILEY L, H!mm NH: RI&O. a MammaEan Origin-Binding Protein, Enhances DNA Bending Neat the Dihydrofolate Reductase Origin of Replication. Mel Cell Brd 1990, 10:623&6243. The 6OkD protein that binds near or in the DHFR origin region was shown to enhance the bend of stably bent DNA near its binding site.
32.
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BURHANS WC, VASSILR, LT, CADDE MS, HEIPSIZ NH, DEPAMPH~~~ ML Identification of an origin of Bidirectional DNA Rep& cation in Mammalian Chromosomes. Cell 1990. 62:955+65. An origin of bidirectional replication was mapped to roughly 450 bp by a new method that can capitalize on the fact that Okazald fragments are only synthesized on the lagging strand. This is the most closely mapped mammalian origin to date.
33. ..
34.
FARACHE G, RABIN SV, R~E~~~~~KI-W~INY J. MORFAU J, TARGA FR, SCHERRER K: Mapping of Structural and TranscriptionRelated Matrix Attachment Sites in the Alpha-GlobI Gene Domain of Avian Etythroblasts and Etythrocytes. Mol Cell Bid 1990,10:53495358.
35.
DIJ~?VEL PA, HAMUN JL Matrix Attachment Regions are Positioned Near Replication lnitiation Sites, Genes and an Interamplicon Junction in the AmpIiIied Dihydrofolate Reductase Domain of CHO Cells. Mel Cell Bid 1988, 8:5398-5409.
210:587-599.
25.
factors
A protein that recognizes the (ATT), rectional origin in the DHFR domain cofractionates with a helicase activity
Inhibition of DNA HI by Distamycin.
Artificial AT-rich constructs and Drmophilu SARs are examined for scaf fold binding and interaction with histone Ill. Rigid DNA with narrow minor-groove structure (e.g. dA, ,T, ,) has higher affinity than altemating dAT. Both SAPS and artificial constructs can he competed by distamycin.
orinins,
VAUGHN JP, DIJhw’rzL PA, HAMIJN JL Replication Initiates In a Broad Zone in the Amplitied CHO Dihydrofolate Reductase Domain. Cell 1990. 6I:IO75Io87. The two-dimensional gel electrophoresis system was used to map replication origins in the amplified DHFR domain. A broad region covering nearly 28 kh contains structures that typify the origin bubble, suggesting that DNA replication initiates here at many sites.
36.
..
VALICHN JP, DIJKWEL PA MUUENIIERS IHF, HM~LLN JL Replication Forks are Associated with the Nuclear Mattix. Nucleic Acids Res Im. 18;1365-1969. Newly synthesized DNA was shown by two-dimensional gel electrophoresis to be enriched in the nuclear matrix fraction prepared by various extraction procedures. This rules out the criticism of high-salt artefacts in earlier studies and provides a way to enrich for repllcative intermediates in mammalian nuclei for twodimensional gel analysis. 37. .
38.
MIUS AD, BLOW JJ, WHITE JG, AMOS WB. WILCOCK D, LWCEY RA: Replication Occurs at Discrete Foci Spaced Throughout Nuclei Replicating In Vitro. J Cell Sci 1989, %:471-477.
39.
NAKAYASU H, BERFZNEY R: Mapping Replicational Sites Eucatyotic CelI Nucleus. J Cell Biol 1989, 108:1-11.
40. .
Brow
JJ. SLEEMAN AM: Replication
pus Egg Extract is Dependent Sci 1990, 95:383391.
In a Xenopus extract/nuclear reconstituted nuclei perform branes and the use of mitotic and DNA replication in vitro
in the
of Purified DNA in Xeno on Nuclear Assembly. J Cell
reconstitution assay it is shown that only DNA replication. The depletion of memextracts eliminate nuclear reconstitution
41. .
&NO GH, the Timing
&K!zY RA: The Nuclear Membrane Determines of DNA Replication in Xenopus Egg Extracts. J Cell Biol 1991, 112:557-566. Chick erythrocyte nuclei form ‘multinuclear aggregates’ in cell-free Xenopus extracts. Such nuclei lack their individual nuclear membranes but share a perimeter envelope. These nuclei undergo DNA replication synchronously at foci distributed throughout the aggregate. 42.
DINGMAN CW Bidirectional Topological Considerations.
SM Gasser, Swiss Institute 1066 Epalinges s/Iausanne,
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