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.

In Schizosaccharomyces pombe, the catalytic subunit of DNA polymerase epsilon (Pol ) is encoded by cdc20 ؉ and is essential for chromosomal DNA replication. Here we demonstrate that the N-terminal half of Pol that includes the highly conserved polymerase and exonuclease domains is dispensable for cell viability, similar to observations made with regard to Saccharomyces cerevisiae. However, unlike budding yeast, we find that fission yeast cells lacking the N terminus of Pol (cdc20 ⌬N-term ) are hypersensitive to DNA-damaging agents and have a cell cycle delay. Moreover, the viability of cdc20 ⌬N-term cells is dependent on expression of rad3 ؉ , hus1 ؉ , and chk1 ؉ , three genes essential for the DNA damage checkpoint control. These data suggest that in the absence of the N terminus of Pol , cells accumulate DNA damage that must be repaired prior to mitosis. Our observation that S phase occurs more slowly for cdc20⌬N-term cells suggests that DNA damage might result from defects in DNA synthesis. We hypothesize that the C-terminal half of Pol is required for assembly of the replicative complex at the onset of S phase. This unique and essential function of the C terminus is preserved in the absence of the N-terminal catalytic domains, suggesting that the C terminus can interact with and recruit other DNA polymerases to the site of initiation.

Chromosome breakage as a result of replication stress has been hypothesized to be the direct consequence of defective replication fork progression, or “collapsed” replication forks. However, direct and genome-wide evidence that collapsed replication forks give rise to chromosome breakage is still lacking. Previously we showed that a yeast replication checkpoint mutant mec1-1, after transient exposure to replication impediment imposed by hydroxyurea (HU), failed to complete DNA replication, accumulated single-stranded DNA (ssDNA) at the replication forks, and fragmented its chromosomes. In this study, by following replication fork progression genome-wide via ssDNA detection and by direct mapping of chromosome breakage after HU exposure, we have tested the hypothesis that the chromosome breakage in mec1 cells occurs at collapsed replication forks. We demonstrate that sites of chromosome breakage indeed correlate with replication fork locations. Moreover, ssDNA can be detected prior to chromosome breakage, suggesting that ssDNA accumulation is the common precursor to double strand breaks at collapsed replication forks.