Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication

Abstract

We report a genome-wide analysis of single-stranded DNA formation during DNA replication in wild type and checkpoint-deficient rad53 yeast cells in the presence of hydroxyurea. In wild type cells, ssDNA first appears at a subset of replication origins and later “migrates” bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA appears at virtually every known origin, but remains there over time, suggesting that replication forks stall. Telomeric regions appear to be especially sensitive to the loss of Rad53 checkpoint function. We also mapped replication origins in Schizosaccharomyces pombe using our method.

Genome-wide characterization of fission yeast DNA replication origins

“…Rad3 acts upstream of the S-phase checkpoint mutant cds1 (Xu et al, 2006), which was used by Feng et al (2006) in origin mapping experiments. We found that in the absence of the Rad3 dependent S-phase checkpoint, only two additional peaks were identified and only eight origins that generally replicated late in S-phase (79-85 min) out of the 401 origins (2%) were significantly induced (between 0.07 and 0.16 increases in hybridization ratios).…”

“…To investigate the possibility that inhibition of late firing origins by the S-phase checkpoint could interfere with origin identification (Feng et al, 2006), the HU experiment was repeated with a checkpoint deficient strain lacking the rad3 gene (Bentley et al, 1996) (Figure 3). Rad3 acts upstream of the S-phase checkpoint mutant cds1 (Xu et al, 2006), which was used by Feng et al (2006) in origin mapping experiments.…”

“…Budding yeast and fission yeast origin sequences are not always interchangeable (Beach and Nurse, 1981;Clyne and Kelly, 1995), suggesting that sequence requirements for origin function can differ between the two yeasts. A recent genome-wide analysis identified 321 origins in fission yeast at a resolution of 12 kb by mapping singlestranded DNA on ORF microarrays in the presence of hydroxyurea (HU) in an S-phase checkpoint deficient strain (Feng et al, 2006). However, only a subset of these (61%) could be identified in cells undergoing a normal S-phase suggesting that an S-phase checkpoint might suppress many origins.…”

“…In budding yeast comprehensive global analyses have identified B260-420 origins in the genome (Macalpine and Bell, 2005;Feng et al, 2006); these are B150 bp DNA fragments containing an 11-17 bp consensus sequence (Theis and Newlon, 1997) reminiscent of viral and bacterial origins. Metazoan origins lack a consensus sequence and often contain cis-regulatory elements embedded in complex genomes (DePamphilis, 2005), which has made it difficult to identify such origins on a genome-wide scale and at the resolution required to investigate origin utilization.…”

Chk1–cyclin A/Cdk1 axis regulates origin firing programs in mammals

“…However, while cis-acting origin of the replication sequence is clearly defined in prokaryotes, it is ambiguous in eukaryotes. For example, a recent whole-genome analysis using HU revealed an S phase checkpoint in budding yeast that suppresses many origins characterized as late (Feng et al 2006;Raveendranathan et al 2006). However, only a few origins fire late during S phase, and the Rad3-dependent S phase checkpoint has little effect on which origins are fired in fission yeast (Hayashi et al 2007;Heichinger et al 2006).…”