COMMENTARY

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Where it all starts: eukaryotic origins of DNA replication

Anja-Katrin Bielinsky* and Susan A. Gerbi
Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA
(e-mail: [email protected]; [email protected]) *Present address: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA

Journal of Cell Science 114, 643-651 The Company of Biologists Ltd

Summary Chromosomal origins of DNA replication in eukaryotic cells not only are crucial for understanding the basic process of DNA duplication but also provide a tool to analyze how cell cycle regulators are linked to the replication machinery. During the past decade much progress has been made in identifying replication origins in eukaryotic genomes. More recently, replication initiation point (RIP) mapping has allowed us to detect start sites for DNA synthesis at the nucleotide level and thus to monitor replication initiation events at the origin very precisely. Beyond giving us the precise positions of start sites, the application of RIP mapping in yeast and human cells has

revealed a single, dened start point at which replication initiates, a scenario very reminiscent of transcription initiation. More importantly, studies in yeast have shown that the binding site for the initiator, the origin recognition complex (ORC), lies immediately adjacent to the replication start point, which suggests that ORC directs the initiation machinery to a distinct site. Therefore, in our pursuit of identifying ORC-binding sites in higher eukaryotes, RIP mapping may lead the way.
Key words: DNA replication, Eukaryotic origins, RIP mapping, ORC binding, Cell cycle control

Introduction At the onset of S phase, eukaryotic cells initiate DNA replication at specic sites, origins of DNA replication, that are found throughout the genome. How cis- and trans-acting regulators interact to promote the initiation of DNA replication is best understood in the budding yeast Saccharomyces cerevisiae. DNA sequences important for replication were rst identied in yeast by ARS (autonomously replicating sequence) assays that determine whether a given DNA fragment initiates replication when placed on a plasmid (Stinchcomb et al., 1979). DNA sequences that promote replication are called replicators. ARS elements were subsequently further divided into functional modules described as A and B domains (Celniker et al., 1984; Walker et al., 1990; Marahrens and Stillman, 1992; Rao et al., 1994; Theis and Newlon, 1994; Huang and Kowalski, 1996; Newlon, 1996). Among all ARS elements isolated to date, domain A comprises the ARS consensus sequence (ACS) (Palzkill and Newlon, 1988; Newlon and Theis, 1993), which is necessary but not sufficient for replication function (Marahrens and Stillman, 1992). The ACS turned out to be recognized in an ATP-dependent manner by a central trans-acting player in replication initiation (Bell and Stillman, 1992; Klemm et al., 1997; Lee and Bell, 1997): the 6-subunit origin recognition complex (ORC), which is probably the eukaryotic counterpart of prokaryotic and viral initiator proteins (e.g. SV40 T-antigen). Initiator proteins were rst proposed by Jacob and coworkers to be essential trans-acting factors for the initiation of DNA replication (Jacob et al., 1964). In general, the initiator fullls three basic functions: (1) it helps to unwind the origin by using an intrinsic or associated helicase (e.g. in eukaryotic

cells, ORC is the initiator and is associated with minichromosome maintenance (Mcm) proteins that are likely to serve as a replicative helicase; see below); (2) it guides the replication machinery to the origin; and (3) in eukaryotic cells it links cell cycle control to origin activation (ORC recruits the cell cycle-regulated Cdc6p to the origin; see below). Unlike in prokaryotes (Jacob et al., 1964; Kornberg and Baker, 1992), in eukaryotic cells the initiator-origin interaction is not sufficient to trigger the initiation of DNA replication. An intermediate licensing step is required to establish replication competence. In yeast, replication competence is accomplished during G1 phase, when the cyclin-dependent kinase Cdk1 activity is low (Fig. 1), by the stepwise assembly of the pre-replication complex (pre-RC) (Diffley et al., 1994; Aparicio et al., 1997; Donovan et al., 1997; Tanaka et al., 1997; Liang and Stillman, 1997; Newlon, 1997; Leatherwood, 1998; Perkins and Diffley, 1998; Weinreich et al., 1999). The budding yeast pre-RC is composed of ORC, Cdc6p and Mcm2p-Mcm7p (Fig. 1), which interact with Mcm10p (Homesley et al., 2000). In Schizosaccharomyces pombe (Nishitani et al., 2000), Drosophila (Whittaker et al., 2000) and Xenopus (Maiorano et al., 2000), an additional factor, Cdt1 (Hofmann and Beach, 1994), is part of the pre-RC and is required for loading of Mcm proteins onto chromatin (Maiorano et al., 2000; Nishitani et al., 2000). At the G1/S phase transition, at which Cdk1 activity levels rise, Cdc45p (Aparicio et al., 1997; Zou and Stillman, 1998; Aparicio et al., 1999; Zou and Stillman, 2000) associates with origins (Diffley, 1998; Donaldson and Blow, 1999). Recently, two groups demonstrated that loading of Cdc45p is dependent on the Cdc7p-Dbf4p kinase (Jares and Blow, 2000; Zou and Stillman, 2000), which is required for origin activation throughout S phase (Bousset and Diffley, 1998; Donaldson et al., 1998).

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Fig. 1. Events leading to origin activation in budding yeast. Origin recognition complex (ORC) binds to an ARS element in yeast (blue rectangle). For the stepwise assembly of the pre-RC during G1 phase when Cdk1p activity is low, ORC recruits Cdc6p, which in turn loads Mcm2p-Mcm7p. When Cdk1p activity rises at the G1/S transition, the pre-RC is disassembled. The post-RC remains stable until the end of mitosis, and owing to high Cdk1p activity the pre-RC cannot re-associate during this time but must await the subsequent G1 phase.

Once replication initiates, the yeast pre-RC is remodeled into the post-RC, which comprises only ORC bound to chromatin (Fig. 1) (Nasmyth, 1996; Diffley, 1998; Donaldson and Blow, 1999). The post-RC is maintained until the end of mitosis, when Cdk1 activity drops and thus helps to prevent reinitiation in the course of one cell cycle (Noton and Diffley, 2000). Only during the subsequent G1 phase is the post-RC retransformed into the pre-RC to license a new round of DNA replication (Fig. 1). In vertebrates, ORC components may dissociate in mitosis and rebind the DNA in G1 phase (Natale et al., 2000, and references therein). In yeast, ORC remains bound to the origin once replication has initiated (Diffley et al., 1994; Aparicio et al., 1997; Tanaka et al., 1997); other individual components of the pre-RC disassemble or become part of the replication fork for example, Mcm2p-Mcm7p (Aparicio et al., 1997; Labib et al., 2000), which are thought to function as a replicative helicase (Kelman et al., 1999; You et al., 1999; Chong et al., 2000; Tye and Sawyer, 2000) and Cdc45p (Aparicio et al., 1997; Tercero and Diffley, 2000). At this point we do not fully understand how the actual replication initiation machinery (e.g. DNA polymerase primase and replication protein A) is recruited to the origin. Recent evidence suggests the involvement of Dpb11p, which interacts genetically with Cdc45p (Masumoto et al., 2000). How components of the initiation complex, such as Mcm proteins or Cdc45p, rearrange to become part of elongating replication forks also remains unclear. However, replication initiation point (RIP) mapping has allowed tracing of the replication products generated by this complicated machine, and we can now paint a picture of initiation events for at least

three cellular origins: the ARS1 element in S. cerevisiae (Bielinsky and Gerbi, 1998; Bielinsky and Gerbi, 1999), the ars1 element in S. pombe (Gomez and Antequera, 1999), and the lamin B2 origin in human cells (Abdurashidova et al., 2000). Complex origins of DNA replication in higher eukarotes The plasmid-based ARS assay has helped to identify numerous replication origins in budding and ssion yeasts (Newlon, 1996; Huberman, 1999). Whereas the elements that drive replication initiation in S. cerevisiae are only ~150 bp in size, their counterparts in S. pombe span 500-1500 bp, lacking an apparent consensus sequence or a modular structure. Higher up the evolutionary ladder, the situation becomes even more extreme. Almost all genomic fragments tested retain ARS activity (when tested in Xenopus egg extract or Drosophila tissue culture cells), and for a while the conclusions drawn from these results favored a sequence-nonspecic, cisindependent mechanism underlying replication initiation in metazoan chromosomes (Coverley and Laskey, 1994; Smith and Calos, 1995; Gilbert, 1998). However, a different picture emerged upon the introduction of physical methods to map origins (positions of DNA synthesis initiation) that enabled replication bubbles (structures emerging upon replication initiation at the origin) to be distinguished from forks (structures indicative of elongating replication forks) (Brewer and Fangman, 1987; Brewer and Fangman, 1991) and determined the direction of fork movement (Huberman et al.,

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Kobayashi et al., 1998). PCR-based assays have also been employed to identify replicators at ectopic sites in the genome, which is an alternative approach to the plasmid-based ARS assay (Aladjem et al., 1998; Malott and Leffak, 1999; Vernis et al., 1999; Tao et al., 2000). Nonetheless, metazoan origins seemed to be much more complex than the small ARS elements in budding yeast. The principle of replication initiation point mapping Despite the progress in mapping origins described above, the question of whether replication initiates at specic sites or anywhere within a given origin be it from yeast or a higher eukaryote remained. The development of RIP mapping (Gerbi and Bielinsky, 1997; Bielinsky and Gerbi, 1998) allowed this question to be answered. RIP mapping takes advantage of the symmetry of a typical replication bubble that emerges once the bidirectionally moving forks have been established (see Fig. 2A) (Gerbi and Bielinsky, 1997; Bielinsky and Gerbi, 1998; Gerbi et al., 1999). To determine the start sites (RIPs) for DNA synthesis, a primer extension assay is employed, using nascent DNA as the template. Nascent strands are hard to distinguish from nicked DNA; early investigations (Okazaki et al., 1968) therefore used selective labeling and digestion procedures to separate nascent from nicked DNA (Okazaki et al., 1975). Similar approaches were used to map start sites of Okazaki fragments at the nucleotide level in eukaryotic viruses (Hay and DePamphilis, 1982; Hendrickson et al., 1987; DePamphilis, 1995), E. coli (Kohara et al., 1985) Fig. 2. Outline of RIP mapping. (A) Nascent DNA (leading strand and Okazaki and bacteriophage lambda (Tsurimoto and fragments) is initiated by a small RNA primer (red rectangles) and used as the Matsubara, 1984). However, these techniques are template in a primer extension reaction (ovals with extending arrows) to not sensitive enough to analyze single-copy origins determine replication initiation points (RIPs). RIP 1 and RIP 1 are called in the chromosomes of eukaryotic cells. transition points (TPs) because they are located at the border between continuous In RIP mapping, sensitivity is enhanced ~1000(leading) and discontinuous (lagging) strand synthesis (Okazaki fragments). fold, and the problem of nicked DNA is overcome (B) Primer extension products (replication intermediates, RIs) are fractionated on by selective degradation of 5 DNA by sequencing gels adjacent to corresponding sequencing lanes. Reactions for the exonuclease (Bielinsky and Gerbi, 1998), which top and bottom strand are shown. The distance between individual RIPs can vary leaves RNA-primed DNA intact (Radding, 1966; from several nucleotides (as in episomal ARS1, Bielinsky and Gerbi, 1998) to Little, 1967). Although, the enzyme is less more than 100 nucleotides (as in the human lamin B2 origin; Abdurashidova et al., 2000). processive on ssDNA (the substrate used in RIP mapping) than on dsDNA, -exonuclease treatment 1987; Huberman, 1997). These two-dimensional gel yields highly puried preparations of nascent strands (Gerbi electrophoresis techniques revealed that metazoan origins were and Bielinsky, 1997; Bielinsky and Gerbi, 1998; Gerbi et al., much larger than origins in budding yeast, surprisingly 1999). Its pH optimum is 9.5 (Radding, 1966; Little, 1967), but heterogeneous in size, ranging from 2 kb to 55 kb (Hamlin and sufficient activity is retained at pH 8.8, which thus avoids Dijkwel, 1995; DePamphilis, 1999), and therefore sometimes alkaline hydrolysis of RNA primers. -exonuclease is also referred to as broad initiation zones. Another origin-mapping effective in other origin-mapping techniques (Aladjem et al., method with similar resolution in the kb range detects the 1998; Kobayashi et al., 1998; Gomez and Antequera, 1999; switch point from leading to lagging strand and conrmed that Abdurashidova et al., 2000; Tao et al., 2000), including those replication in metazoa does not initiate randomly throughout analyzing nascent-strand abundance by quantitative PCR the genome (Hamlin and Dijkwel, 1995; Gerbi et al., 1999). (Giacca et al., 1994; Giacca et al., 1997), and is especially More sensitive approaches that employ PCR to determine useful in systems such as S. cerevisiae that normally do not the abundance of nascent strands (Vassilev and Johnson, 1989; readily take up the density label BrdU. Giacca et al., 1994) rened the picture and allowed origins to In RIP mapping, nascent DNA typically isolated from be mapped to as small a region as 0.5 kb (Giacca et al., 1994; asynchronously growing cells is analyzed by primer extension.

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have also been identied genetically (Newlon, 1996; Huberman, 1999). However, linker scanning mutation analysis, which dened functional domains in budding yeast ARS1 (Marahrens and Stillman, 1992), failed to do so in ssion yeast ars1 (Clyne and Kelly, 1995). This suggested functional redundancy within the 782-bp replicator. In the same vein,

Primers are extended to various initiation points (e.g. RIP 1RIP 3 for the top strand in Fig. 2A), and extension products are fractionated on sequencing gels in the nal step of the analysis (Fig. 2B). The smallest fragment marks the transition point (TP) between leading and lagging strand. As described below, RIP mapping shows that leading strand synthesis starts at a unique site, in both small and large origins. Striking similarities between yeast and human origins RIP mapping has been reported for three different chromosomal origins: ARS1 in S. cerevisiae (Bielinsky and Gerbi, 1999); ars1 in S. pombe (Gomez and Antequera, 1999), and the human lamin B2 origin in HeLa cells (in this case linker-mediated PCR (LM-PCR) was substituted for the primer extension step to increase sensitivity) (Abdurashidova et al., 2000). The initiation patterns of the origins from these three different systems show highly conserved features. This is remarkable considering their size differences and, most importantly, the evolutionary distance between them. The study of chromosomal ARS1 in yeast revealed that leading strand DNA synthesis on the top and bottom strands initiates within a 2bp region anked by the ORC-binding site and the easily unwound domain B2 (see Fig. 3). Further experiments in ligase-I-decient cdc9 mutants suggested that leading strand initiation occurs exclusively at the TP (Bielinsky and Gerbi, 1999); additional initiation points (black arrows in Fig. 3) thus do not represent alternative leading strand start sites. The initiation pattern of chromosomal ARS1 reects a degree of precision that was unexpected, especially given the precedent from eukaryotic viruses for which unwinding of the origin was hypothesized to expose multiple possible start sites, from which one is chosen stochastically (Hay and DePamphilis, 1982; Hendrickson et al., 1987). Another important difference is the relatively low frequency of Okazaki fragment initiation sites (Fig. 3A). Unlike the plasmid copy of ARS1 (Bielinsky and Gerbi, 1998), which shows initiation at much higher frequencies, start sites for Okazaki fragments in the chromosome appear to be evenly spaced by ~40 nucleotides (Bielinsky and Gerbi, 1999). Interestingly, the size distribution of Okazaki fragments in yeast shows two peaks: ~40-50 nucleotides and ~125 nucleotides (Bielinsky and Gerbi, 1999). Studies of cdc9 mutants suggest that the longer fragments are not, as had been proposed (Nethanel and Kaufmann, 1990; Nethanel et al., 1992; Salas et al., 1996), the product of multiple smaller fragments (Bielinsky and Gerbi, 1999). The ars elements in S. pombe (replicators)

Fig. 3. Conguration of three different eukaryotic origins. (A) Distribution of start sites for DNA synthesis (leading strand initiation points denoted by red arrows; lagging strand initiation points denoted as black arrows) in ARS1 of S. cerevisiae (episomal copy, Bielinsky and Gerbi, 1998; chromosomal copy, Bielinsky and Gerbi, 1999), ars1 in S. pombe (Gomez et al., 1999) and the human lamin B2 origin (Abdurashidova et al., 2000) are shown. The origin recognition complex (ORC)binding site in ARS1 (Bell and Stillman, 1992) and a site protected by the originbinding protein during S phase (OBP) in the human lamin B2 origin (Dimitrova et al., 1996; Abdurashidova et al., 1998; Abdurashidova et al., 2000) are depicted. (B) Nucleotide sequence of the regions surrounding the transition points of ARS1 (S. cerevisiae), ars1 (S. pombe) and the human lamin B2 origin. In ARS1, the ORCbinding site (Bell and Stillman, 1992) and domains A, B1 and B2 (Marahrens and Stillman, 1992) are shown. Sequences underlined in red represent matches to the ARS consensus sequence (ACS) (Newlon and Theis, 1993). The site protected by the origin binding protein (OBP) in the human lamin B2 origin is indicated (Dimitrova et al., 1996; Abdurashidova et al., 1998; Abdurashidova et al., 2000)

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deletion of an 11-bp A/T-rich element similar to the ACS had no effect on genomic replication, as did deletion of a region that had a strong effect on plasmid stability (Gomez and Antequera, 1999). Nevertheless, the recently reported initiation pattern of chromosomal ars1 in S. pombe is very similar to that of ARS1 in S. cerevisiae (Fig. 3A). The leading strand start sites for the top and bottom strand lie within a 4-bp region, and the start sites for the rst Okazaki fragments are ~55-60 bp away (Gomez and Antequera, 1999). Given the functional redundancy of subdomains within the S. pombe ars1 replicator (Clyne and Kelly, 1995), the similarities in the initiation patterns of ARS1 and ars1 are surprising and suggest that regulation of replication initiation is signicantly inuenced by the chromosomal context. The chromosomal context probably also causes the differences in the initiation patterns of genomic and episomal ARS1 in budding yeast (Bielinsky and Gerbi, 1998; Bielinsky and Gerbi, 1999). We have learned much about replication initiation events in yeast, but what is the situation in metazoa? One of the bestcharacterized metazoan origins is located at the 3 end of the human lamin B2 gene. This origin was initially mapped by quantitative, competitive PCR and encompasses ~470 bp (Giacca et al., 1994). Moreover, protein-DNA interactions at the origin (Dimitrova et al., 1996) have been shown to be modulated during the cell cycle in a manner very reminiscent of the protein-protection pattern displayed over ARS1 DNA in budding yeast (Abdurashidova et al., 1998). However, a difference is that the lamin B2 origin does not appear to bind any proteins during mitosis (Abdurashidova et al., 1998), whereas ORC remains associated with ARS1 throughout the cell cycle (Diffley et al., 1994; Aparicio et al., 1997; Tanaka et al., 1997). The question of whether the protein-DNA interactions detected at the lamin B2 origin are due to pre-RC (during G1 phase) and post-RC (during S phase) formation remains unanswered. However, recent RIP-mapping analyses (modied by LM-PCR) at this locus place the replication start points close to the site of protein-DNA interaction (Fig. 3A) (Dimitrova et al., 1996; Abdurashidova et al., 1998; Abdurashidova et al., 2000). Overall, the initiation pattern is very symmetric, and Okazaki fragment start sites occur every 50-140 bp (Abdurashidova et al., 2000). Experiments using emetine, a potent protein synthesis inhibitor, which was previously demonstrated to suppress Okazaki fragment synthesis (Burhans et al., 1991), conrmed that replication of the lamin B2 locus starts, as in yeast ARS1, at a single leadingstrand-initiation site (Abdurashidova et al., 2000). Moreover, the start site on the top strand is just 3 bp away from that on the bottom strand. Despite the large evolutionary distance between yeast and man, the precisely regulated choice of replication start sites appears to be highly conserved, at least in the cases studied to date. ORC-origin interaction key to choosing the start site? Transcription and DNA replication benet from an accuracy of start site. However, although the entire replication initiation apparatus and fundamental aspects of the initiation process seem to be evolutionarily conserved (Dutta and Bell, 1997), there appear to be no target sequences in replication that are

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conserved between species, which contrasts with transcription. Neither ars1 in S. pombe (Gomez and Antequera, 1999) nor the human lamin B2 origin (Abdurashidova et al., 2000) has S. cerevisiae-ACS-like sequences close to their replication start sites (Fig. 3B). Furthermore, Orc2p from budding yeast cannot functionally substitute for its vertebrate counterpart (Carpenter et al., 1996). Nevertheless, like ARS elements in budding yeast, origins in higher eukaryotes show a very high AT content and are rich in A/T clusters (Bogan et al., 2000). This suggests that the ACS as a recognition site for ORC in budding yeast has either evolved into a new target sequence in higher eukaryotes, or that the binding between ORC and DNA underwent changes in a co-evolutionary manner. Recent evidence from an analysis of ORC in ssion yeast supports the latter notion. The Orc4p homolog in S. pombe (Orp4p) carries an unusually extended, N-terminal AT-hook motif (Chuang and Kelly, 1999) similar to that typically found in HMG-I (Y) proteins (Aravind and Landsman, 1998). AT-hook domains are known to interact with AT tracts (Maher and Nathans, 1996). HMG-I (Y) proteins usually contain three or four AT-hook motifs (Aravind and Landsman, 1998), and S. pombe Orp1p (Muzi-Falconi and Kelly, 1995) and S. cerevisiae Orc2p (Bell et al., 1995) each contain a single AT-hook motif. Interestingly, S. pombe Orp4p carries nine AT-hook motifs (Chuang and Kelly, 1999). Chuang and Kelly have suggested that, in ssion yeast, ORC recognizes target sites through its AT-hook domain and that its binding affinity depends on how many of the nine AT-hook motifs interact with DNA (Chuang and Kelly, 1999). This hypothesis is consistent with the high AT content of ars elements in S. pombe and is supported by a recent report that demonstrates that the origin activity of ars 2004 depends on the number of A/T stretches and the extent of their clustering (Okuno et al., 1999). Moreover, Orp1p specically associates with the AT-rich regions I and III in ars 2004 throughout the cell cycle (Ogawa et al., 1999). Since extended AT-hook domains are not found in ORC analogs of other species (Dutta and Bell, 1997), ORC binding through the AT-hook motif might be a specialization of S. pombe, particularly given that ars elements in ssion yeast have extremely AT-rich regions (DePamphilis, 1999; Bogan et al., 2000). However, AT tracts are still very common in metazoan origins, and it is possible that ORC-DNA interaction is assisted by AT-hooks of HMG-I (Y) proteins in trans (Chuang and Kelly, 1999). Mizushima et al. have shown that ORC might need assistance in regulating its binding activity: puried Cdc6p from S. cerevisiae enhanced the specicity of ORC-DNA interaction in vitro (Mizushima et al., 2000). It remains to be seen whether Cdc6p and/or possibly other, unidentied factors modulate ORC binding in higher eukaryotes and whether this modulation affects origin choice at the origin decision point in mammalian cells (Wu and Gilbert, 1996; Wu and Gilbert, 1997). A key challenge in the replication eld is to characterize ORC-DNA interactions in other species besides S. cerevisiae. However, progress has been slow, and to date we still know very little about this subject. It is currently unclear whether ORCs of more highly evolved organisms recognize specic sequences or structural features similar to the AT-rich regions in the replicators of S. pombe. The chorion loci in Drosophila melanogaster have proved to be a useful model system for studies into the regulation of replication initiation (Calvi and

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Topological changes monitored at ARS1 during different stages of the G1/S transition implicate the B2 domain as a facilitator for initiation but also demonstrate that ssDNA is rst detected in domain A, which is part of the ORC-binding site (Geraghty et al., 2000). Further topological and RIPmapping studies will help us to dissect the roles of the various cis- and trans-acting players. To sum up, leading strand synthesis initiates at a unique start site, which may be determined by ORC that is bound adjacent to the start site in cases where it has been studied. The larger origins found in metazoa may contain multiple ORC-binding sites and may therefore reect population polymorphism in which leading strand start sites differ between individual replicating DNA molecules. Three-dimensional gel analysis suggests that clustered replication bubbles do not occur on the same molecule (Liang and Gerbi, 1994). As for the lagging strand (Okazaki fragment) start sites, they do not share any consensus sequence, even within a given locus, and the mechanism underlying their regular spacing remains to be claried. Conclusions The initiation of DNA replication is a highly regulated, very precisely executed process in eukaryotic cells. To date, evidence suggests that replication, like transcription, starts from a single dened site in yeast and in humans. Since we have only few examples of initiation patterns at nucleotide resolution, this relatively simple notion may be challenged as our understanding of metazoan origins and their regulation progresses. The controversy of small versus broad origins requires resolution, but it might turn out that what we today call broad zones may well be clusters of smaller origins that operate in the way outlined here. The nature of ORC-DNA interaction, and cell-cycledependent ORC control, will be central to understanding of the regulation of metazoan origin use during development. In Xenopus embryos, initiation of DNA synthesis appears to be sequence independent but later, at the mid-blastula transition, becomes specically linked to replication origins. The underlying basis for these changes might be the differentially controlled association of ORC with DNA during development which could, for example, depend on changes in ORC abundance, ORC modication and/or the requirement for regulatory co-factors. Finally, a detailed characterization of replication origins in higher eukaryotes might also help to elucidate the relationship between DNA replication and other DNA-metabolic processes, such as DNA repair, transcription and chromatin remodeling.
The authors were supported by The Krause Family Special Fellowship of the Leukemia and Lymphoma Society (A.-K.B.) and NIH GM35929 (S.A.G.).

Spradling, 1999). These loci undergo repeated rounds of DNA replication as part of a developmental program to provide extra copies of chorion genes as templates for transcription. The onset of DNA amplication of chorion genes is very likely to employ the same initiation machinery (Landis et al., 1997; Landis and Tower, 1999) and regulators (Calvi et al., 1998; Royzman et al., 1999) that play key roles in mitotic replication. The mechanism by which the iron rule of initiating DNA replication once and only once per cell cycle is overridden remains obscure. However, recent in vitro studies demonstrate that ORC binds to the amplication control element 3 and to ori , both of which lie in the general region of initiation (Austin et al., 1999), as detected previously by twodimensional gels (Delidakis and Kafatos, 1989; Heck and Spradling, 1990). The initiation pattern of the chorion origin has not yet been determined at the nucleotide level. Therefore, the relationship between the replication start sites and the ORC-binding sites remains to be illuminated. In another dipteran y, Sciara coprophila, which also exhibits developmentally regulated amplication of DNA (Gerbi et al., 1993), we have detected ORC binding (Bielinsky et al., submitted) to the amplication origin of DNA puff II/9A (Liang et al., 1993; Liang and Gerbi, 1994). The ORC-binding site maps very close to the replication start site (Bielinsky et al., submitted), extending the yeast paradigm to higher eukaryotes. A candidate for a human ORC-binding site is the 74-bp region protected by the origin-binding protein (OBP) at the human lamin B2 origin (Fig. 3B) (Dimitrova et al., 1996). Although HoxC10p and HoxC13p can bind to the 74-bp region (de Stanchina et al., 2000), it is unclear whether ORC occupies this site as well. OBP seems to be displaced during mitosis (Abdurashidova et al., 1998). Indeed, Natale et al. recently demonstrated that mammalian ORC partly disassembles during mitosis: in hamster, Orc1p associates and disassociates from chromatin in a cell-cycle-regulated fashion (Natale et al., 2000). However, at least one subunit, Orc2p, seems to stay chromatin bound throughout the cell cycle in hamster (Natale et al., 2000) and human cells (Ritzi et al., 1998). In contrast, experiments in Xenopus egg extract suggest that ORC is displaced once licensing has occurred in late G1 phase (Hua and Newport, 1998; Rowles et al., 1999). Although the fate of ORC needs further investigation, accumulating evidence indicates that, in higher eukaryotes, similarly to yeast, replication is initiated close to the ORCbinding site (Bielinsky et al., submitted). Even if ORC binding is modulated by cis- and trans-acting factors different from those known to act in yeast, ORC might be the determining factor as to the choice of replication start site, by providing a scaffold for the initiation machinery. ORC binding itself might expose to the initiation machinery that stretch of DNA that is subsequently used as the template for the leading strand. In this context, note that, upon post-RC formation at the G1/S transition, the region conned to the area surrounding the transition point in ARS1 of budding yeast exhibits strong DNase I hypersensitivity (Diffley and Cocker, 1992). However, it is also possible that the initiation machinery, although recruited close to the ORC-binding site, acts at a site primarily determined by the location of a DNAunwinding element, such as the B2 domain in yeast ARS1 (Huang and Kowalski, 1993; Lin and Kowalski, 1997).

References
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