Completion of Replication Map ofSaccharomyces cerevisiaeChromosome III

Abstract

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...

Behavior of replication origins in Eukaryota – spatio-temporal dynamics of licensing and firing

“…The timing profile shows distribution of activation time along the chromosome -from green parts replicated in about 10 min to gray regions that are replicated in late subperiods of the S phase. The timing profile seems to almost perfectly reflect the activity and efficiency of particular origins (according to Raghuraman et al 67 and Poloumienko et al 113 , modified).…”

Unique Error Signature of the Four-subunit Yeast DNA Polymerase ϵ

“…Toward this long term goal, the present study compares the spectrum of mutations generated by Pol ⑀ in vitro with the spectrum of mutations that result from spontaneous DNA replication errors in vivo in a region ϳ4,400 base pairs from the ARS306 replication origin that fires early in S phase (66). The data in Table IV reveal that the spectra of spontaneous mutations in the URA3 reporter gene in a pol2-4 msh6 genetic background are significantly different for the two orientations of the reporter gene.…”

Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability inSaccharomyces cerevisiae

“…replication in only 10% of cell cycles (Poloumienko et al 2001). Therefore under replication stress, a fork proceeding from ARS 310 permits hairpin formation on the lagging strand when it reaches FS2, and cleavage of this hairpin generates a one-end double-strand break (Figure 7).…”

“…The replication origin closest to FS2 is ARS 310, which is located 1 kb centromere-proximal to the fragile site. This origin initiates replication relatively early in 90% of cell cycles (Poloumienko et al 2001). Centromere-distal to FS2, the nearest highly efficient replication origin is ARS 315, located 55 kb away; and ARS 313, located 25 kb distal, initiates replication in only 10% of cell cycles (Poloumienko et al 2001).…”

“…This origin initiates replication relatively early in 90% of cell cycles (Poloumienko et al 2001). Centromere-distal to FS2, the nearest highly efficient replication origin is ARS 315, located 55 kb away; and ARS 313, located 25 kb distal, initiates replication in only 10% of cell cycles (Poloumienko et al 2001). Therefore under replication stress, a fork proceeding from ARS 310 permits hairpin formation on the lagging strand when it reaches FS2, and cleavage of this hairpin generates a one-end double-strand break (Figure 7).…”