Analysis of Replication Origin Function on Chromosome III of Saccharomyces cerevisiae

Abstract

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

Saccharomyces Rrm3p, a 5′ to 3′ DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA

“…In wild-type cells, even longer exposure of the gel in Figure 5A (left panel) did not reveal bubble-shaped intermediates, consistent with the finding that this ARS is not active in wild-type cells (Newlon et al 1993). However, bubble-shaped intermediates were visible in rrm3⌬ DNA (Fig.…”

“…The left telomere of Chromosome III has X but no YЈ DNA (Button and Astell 1986). Although the X has an ARS, the ARS is inactive in wild-type cells (Newlon et al 1993), such that replication of the terminal SphI fragment occurs by leftwardmoving forks (ARS is marked with an asterisk in Fig. 5A).…”

Checkpoint-mediated control of replisome–fork association and signalling in response to replication pausing

“…ChIP analysis was carried out on the chromosomal region containing ARS305 (Newlon et al, 1993), which has been extensively characterized at the level of firing and processing in the presence of HU both in wt-and checkpoint-defective mutants (Lopes et al, 2001). Besides the fragment containing ARS305, we have also analysed three regions positioned 9 kilobases (kb) to the left and 8 and 17 kb to the right, respectively, of ARS305 (Figure 1a).…”

Transcription-associated mutagenesis in yeast is directly proportional to the level of gene expression and influenced by the direction of DNA replication

“…Plasmids pCM225 and pCM244 used in the construction of the pTET reporter system were acquired from Euroscarf and are described in Belli et al [22]. The strains with the doxycycline (Dox)-regulated lys2ΔA746 allele (pTET-lys2ΔA746) in both orientations relative to an origin of replication on Chromosome III, ARS306 [23], were constructed by the following steps (specific details are available upon request). First, the endogenous LYS2 gene on chromosome II was replaced with a PCR-generated fragment conferring hygromycin resistance (lys2Δ∷hyg).…”