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.

Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

Hydroxyurea (HU) is a DNA replication inhibitor that negatively affects both the elongation and initiation phases of replication and triggers the "intra-S phase checkpoint." Previous work with budding yeast has shown that, during a short exposure to HU, MEC1/RAD53 prevent initiation at some late S phase origins. In this study, we have performed microarray experiments to follow the fate of all origins over an extended exposure to HU. We show that the genome-wide progression of DNA synthesis, including origin activation, follows the same pattern in the presence of HU as in its absence, although the time frames are very different. We find no evidence for a specific effect that excludes initiation from late origins. Rather, HU causes S phase to proceed in slow motion; all temporal classes of origins are affected, but the order in which they become active is maintained. We propose a revised model for the checkpoint response to HU that accounts for the continued but slowed pace of the temporal program of origin activation.

Temporal regulation of origin activation is widely thought to explain the pattern of early-and latereplicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5D strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins (''late'' origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times.