In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ϳ40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements on S. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in Յ10% of cells in the population and two ARS elements active in Ն90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome. INTRODUCTIONThe replication of eukaryotic chromosomes initiates at multiple replication origins spaced at intervals of 40 -100 kb. In the budding yeast, Saccharomyces cerevisiae, replication origins depend upon cis-acting replicators called autonomously replicating sequence (ARS) elements, which are recognized by their ability to maintain extrachromosomal plasmids. The initiation of replication at individual replicators is a tightly regulated process. Replication initiations are confined to S phase, and individual replicators initiate at reproducible times during S phase, some early and some late (Reynolds et al., 1989, and references therein;Friedman et al., 1997;Donaldson et al., 1998b). Moreover, the efficiencies of initiation vary from one replicator to another, the extreme case being ARS elements that are not active as replication origins in their normal chromosomal positions (Dubey et al., 1991;Friedman et al., 1997;Yamashita et al., 1997).Replicators become competent to initiate replication during the G1 phase of the cell cycle through the stepwise assembly of prereplicative complexes on replicators that are bound by origin recognition complex (ORC) (reviewed by Dutta and Bell, 1997). The actual initiation events require the activities of at least two protein kinases, the cyclin-dependent kinase (CDK) Cdc28p associated with cyclin B (Clb5p or Clb6p) and the Cdc7p kinase associated with its regulatory subunit Dbf4p. The assembly of prereplicative complexes is prevented by the activity of cyclin B-associated CDK, effectively preventing reinitiation at origins during a single S phase (reviewed by Diffley, 1996). Timing determinants also appear to be specified during G1 of the cell cycle (Raghuraman et al., 1997), although the relationship between establishment of the prereplication complex and the specification of initiation timing is unclear. In the case of a latereplicating region of chromosome XIV, DN...

Eukaryotic chromosomes contain long linear DNA molecules that initiate replication at multiple sites. Although the chromosomal DNA molecules of the yeast Saccharomyces cerevisiae are two to three orders of magnitude smaller than those of multicellular eukaryotes, their replication pattern is similar, with active origins spaced at approximately 40-kb intervals (for review, see Newlon 1988) and a regular temporal order of replication (for review, see Fangman and Brewer 1991). An important advantage of yeast in the study of DNA replication is the ability to identify potential chromosomal origins of replication by a simple plasmid assay. These autonomously replicating sequence (ARS) elements act in cis to promote high-frequency transformation and the extrachromosomal maintenance of plasmids (for review, see Campbell and New-Ion 1991).Our analysis of ARS elements in the 200-kb region of chromosome III between the left telomere and the MAT locus in the middle of the right arm (Newlon et al. 1986) raised a number of questions. Although they are not uniformly distributed, the average spacing between the 14 ARS elements identified is approximately 14 kb, only one-third of the average spacing between active replication origins. This shorter spacing could reflect a higher density of origins on chromosome III than in the genome as a whole. Alternatively, some ARS elements might not be active as chromosomal replication origins, or potential replication origins might not initiate replication in every cell cycle. Even the average density of active replication origins seems considerably higher than necessary. Based on measured rates of fork movement and the average length of S phase, a single bidirectional replication origin that initiates early in S phase should be able to replicate between 120 kb and 360 kb of DNA (for review, see Newlon 1988). A high density of origins could be required if there were replication fork barriers along the chromosome or if replication of the chromosome initiated late in S phase. Alternatively, replication origins might be redundant under normal laboratory growth conditions. As an approach to understanding these questions, we have undertaken a systematic study

The observed spacing between chromosomal DNA replication origins in Saccharomyces cerevisiae is at least four times shorter than should be necessary to ensure complete replication of chromosomal DNA during the S phase. To test whether all replication origins are required for normal chromosome stability, the loss rates of derivatives of chromosome III from which one or more origins had been deleted were measured. In the case of a 61-kb circular derivative of the chromosome that has two highly active origins and one origin that initiates only 10 to 20%o of the time, deletion of either highly active origin increased its rate of loss two-to fourfold. Deletion of both highly active origins caused the ring chromosome to be lost in approximately 20%o of cell divisions. This very high rate of loss demonstrates that there are no efficient cryptic origins on the ring chromosome that are capable of ensuring its replication in the absence of the origins that are normally used. Deletion of the same two origins from the full-length chromosome II, which contains more than six replication origins, had no effect on its rate of loss. These results suggest that the increase in the rate of loss of the small circular chromosome from which a single highly active origin was deleted was caused by the failure of the remaining highly active origin to initiate replication in a small fraction (approximately 0.003) of cell cycles.Chromosomal sequences that enable plasmids to replicate in the nucleus of the yeast Saccharomyces cerevisiae have been identified. These have been termed autonomously replicating sequence or ARS elements. Fourteen ARS elements have been identified and mapped in a 200-kb region of chromosome III (4, 27, 28). By two-dimensional (2-D) gel electrophoresis techniques, 10 of the chromosome III ARS elements have been examined directly for chromosomal origin function. Replication origins have been mapped to within a few hundred base pairs of six of these ARS elements: ARS305 (7, 9, 17); ARS306 (7, 48); ARS307, ARS308, and ARS309 (10); and ARS310 (6). The other four ARS elements tested, ARS301, ARS302, ARS303 (9), and ARS304 (5), do not function as chromosomal origins at detectable levels (Fig. 1).ARS elements are essential for chromosomal origin function. Deletion of a 200-bp fragment containing ARS306 and point mutations in domain A of ARS307 that abolish ARS activity also abolish chromosomal origin function when they are placed in their natural chromosomal context (7). In addition, multiple point mutations within the consensus sequence of the HMR E ARS also abolish the associated chromosomal replication origin function (33).On yeast chromosomes, adjacent replication origins have an average spacing of 36 kb (26). A calculation based on reported fork rates (19,32,34) and an S phase of 25 min at 30°C (46) suggests that between 120 and 300 kb of chromosomal DNA could be replicated from a single replication origin if it initiated early in the S phase. Thus, the spacing between replication origins on yeast chromosomes appears ...

Replication origins in Saccharomyces cerevisiae are spaced at intervals of approximately 40 kb. However, both measurements of replication fork rate and studies of hypomorphic alleles of genes encoding replication initiation proteins suggest the question of whether replication origins are more closely spaced than should be required. We approached this question by systematically deleting replicators from chromosome III. The first significant increase in loss rate detected for the 315-kb full-length chromosome occurred only after all five efficient chromosomal replicators in the left two-thirds of the chromosome (ARS305, ARS306, ARS307, ARS309, and ARS310) had been deleted. The removal of the inefficient replicator ARS308 from this originless region caused little or no additional increase in loss rate. Chromosome fragmentations that removed the normally inactive replicators on the left end of the chromosome or the replicators distal to ARS310 on the right arm showed that both groups of replicators contribute significantly to the maintenance of the originless chromosome. Surprisingly, a 142-kb derivative of chromosome III, lacking all sequences that function as autonomously replicating sequence elements in plasmids, replicated and segregated properly 97% of the time. Both the replication initiation protein ORC and telomeres or a linear topology were required for the maintenance of chromosome fragments lacking replicators.In eukaryotes, DNA replication initiates at specific sites called replication origins. cis-acting sequences called replicators define the positions and regulate the activity of replication origins by promoting the assembly of prereplicative complexes (pre-RCs) during the G 1 phase of the cell cycle. Eukaryotic replicators were first identified in the budding yeast Saccharomyces cerevisiae on the basis of their ability to promote the extrachromosomal maintenance of plasmids (22, 50). The dissection of these autonomously replicating sequence (ARS) elements revealed an essential 11-bp sequence, called the ARS consensus sequence (ACS), that is required for both plasmid and chromosomal replicator activity (reviewed in reference 34). The ACS is the core of the binding site for the highly conserved six-subunit origin recognition complex (ORC), which recruits and assembles the other components of the pre-RC (reviewed in reference 1). Like the ORC, the other components of the pre-RC are highly conserved throughout the eukaryotic kingdom.Upon entry into S phase, pre-RCs are activated to initiate replication according to a temporal program whose determinants are poorly understood. The activation of replication origins requires the activity of two kinases, a cyclin-dependent kinase composed of the catalytic subunit encoded by CDC28 and regulatory subunits encoded by CLB5 and CLB6, and the Dbf4-dependent kinase, composed of a catalytic subunit encoded by CDC7 and a regulatory subunit encoded by DBF4. During replication initiation, DNA is unwound at origins and replication fork proteins are assembled to form repl...

In eukaryotic chromosomes, DNA replication initiates at multiple origins. Large inter-origin gaps arise when several adjacent origins fail to fire. Little is known about how cells cope with this situation. We created a derivative of Saccharomyces cerevisiae chromosome III lacking all efficient origins, the 5ORIΔ-ΔR fragment, as a model for chromosomes with large inter-origin gaps. We used this construct in a modified synthetic genetic array screen to identify genes whose products facilitate replication of long inter-origin gaps. Genes identified are enriched in components of the DNA damage and replication stress signaling pathways. Mrc1p is activated by replication stress and mediates transduction of the replication stress signal to downstream proteins; however, the response-defective mrc1AQ allele did not affect 5ORIΔ-ΔR fragment maintenance, indicating that this pathway does not contribute to its stability. Deletions of genes encoding the DNA-damage-specific mediator, Rad9p, and several components shared between the two signaling pathways preferentially destabilized the 5ORIΔ-ΔR fragment, implicating the DNA damage response pathway in its maintenance. We found unexpected differences between contributions of components of the DNA damage response pathway to maintenance of ORIΔ chromosome derivatives and their contributions to DNA repair. Of the effector kinases encoded by RAD53 and CHK1, Chk1p appears to be more important in wild-type cells for reducing chromosomal instability caused by origin depletion, while Rad53p becomes important in the absence of Chk1p. In contrast, RAD53 plays a more important role than CHK1 in cell survival and replication fork stability following treatment with DNA damaging agents and hydroxyurea. Maintenance of ORIΔ chromosomes does not depend on homologous recombination. These observations suggest that a DNA-damage-independent mechanism enhances ORIΔ chromosome stability. Thus, components of the DNA damage response pathway contribute to genome stability, not simply by detecting and responding to DNA template damage, but also by facilitating replication of large inter-origin gaps.

We have tested the clones used in the European Yeast Chromosome III Sequencing Programme for possible artefacts that might have been introduced during cloning or passage through Escherichia coli. Southern analysis was performed to compare the BamHI, EcoRI, HindIII and PstI restriction pattern for each clone with that of the corresponding locus on chromosome III in the parental yeast strain. In addition, further enzymes were used to compare the restriction maps of most clones with the map predicted by the nucleotide sequence (Oliver et al., 1992). Only four of 506 6-bp restriction sites predicted by the sequence were not observed experimentally. No significant cloning artefacts appear to disrupt the published sequence of chromosome III. The restriction patterns of six yeast strains have also been compared. In addition to two previously identified sites of Ty integration on chromosome III (Warmington et al., 1986; Stucka et al., 1989; Newlon et al., 1991), a new polymorphic site involving Ty retrotransposition (the Far Right-Arm transposition Hot-Spot, FRAHS) has been identified close to CRY1. On the basis of simple restriction polymorphisms, the strains S288C, AB972 and W303-1b are closely related, while XJ24-24a and J178 are more distant relatives of S288C. A polyploid distillery yeast is heterozygous for many polymorphisms, particularly on the right arm of the chromosome.

Eukaryotic chromosomes are duplicated during S phase and transmitted to progeny during mitosis with high fidelity. Chromosome duplication is controlled at the level of replication initiation, which occurs at cis-acting replicator sequences that are spaced at intervals of $40 kb along the chromosomes of the budding yeast Saccharomyces cerevisiae. Surprisingly, we found that derivatives of yeast chromosome III that lack known replicators were replicated and segregated properly in at least 96% of cell divisions. To gain insight into the mechanisms that maintain these ''originless'' chromosome fragments, we screened for mutants defective in the maintenance of an ''originless'' chromosome fragment, but proficient in the maintenance of the same fragment that carries its normal complement of replicators (originless fragment maintenance mutants, or ofm). We show that three of these Ofm mutations appear to disrupt different processes involved in chromosome transmission. The OFM1-1 mutant seems to disrupt an alternative initiation mechanism, and the ofm6 mutant appears to be defective in replication fork progression. ofm14 is an allele of RAD9, which is required for the activation of the DNA damage checkpoint, suggesting that this checkpoint plays a key role in the maintenance of the ''originless'' fragment.

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