Checkpoint-independent scaling of the Saccharomyces cerevisiaeDNA replication program

Abstract

BackgroundIn budding yeast, perturbations that prolong S phase lead to a proportionate delay in the activation times of most origins. The DNA replication checkpoint was implicated in this scaling phenotype, as an intact checkpoint was shown to be required for the delayed activation of late origins in response to hydroxyurea treatment. In support of that, scaling is lost in cells deleted of mrc1, a mediator of the replication checkpoint signal. Mrc1p, however, also plays a role in normal replication.ResultsTo examine whether the replication checkpoint is required for scaling the replication profile with S phase duration we measured the genome-wide replication profile of different MRC1 alleles that separate its checkpoint function from its role in normal replication, and further analyzed the replication profiles of S phase mutants that are checkpoint deficient. We found that the checkpoint is not required for scaling; rather the unique replication phenotype of mrc1 deleted cells is attributed to the role of Mrc1 in normal replication. This is further supported by the replication profiles of tof1Δ which functions together with Mrc1p in normal replication, and by the distinct replication profiles of specific POL2 alleles which differ in their interaction with Mrc1p.ConclusionsWe suggest that the slow fork progression in mrc1 deleted cells reduces the likelihood of passive replication leading to the activation of origins that remain mostly dormant in wild-type cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0079-z) contains supplementary material, which is available to authorized users.

Checkpoint-Independent Regulation of Origin Firing by Mrc1 through Interaction with Hsk1 Kinase

“…Interestingly, this stimulation was not observed for mrc1-3A (22). Furthermore, previous reports suggest the presence of checkpoint-independent scaling of origin firing mediated by Mrc1 (19,20). These results point to the possibility that Mrc1 plays an unknown but novel role in the regulation of early origin firing.…”

“…Mrc1 was reported previously to be required for the scaling of origin activation in budding yeast, and this role of Mrc1 was shown to be checkpoint independent (19,20). We speculate that mechanisms involving the Mrc1-Hsk1 interaction may impose similar regulation of origin firing in fission yeast as well, since the ΔHBS mutant exhibits precocious firing of selective early-firing origins.…”

“…It has been implicated in efficient replication fork progression (28)(29)(30). It was also reported that the checkpoint-independent function of Mrc1 is involved in the scaling of origin activation in slowed S-phase progression (19,20). We previously reported the possibility that Mrc1 regulates the firing of some early-firing origins in a checkpoint-independent manner (22).…”

“…In contrast, early-firing origins fire early, even in the extended S phase, in ΔMRC1 cells (19). Importantly, this requirement of Mrc1 for the scaling of origin activation could still be mediated by a checkpoint-deficient mutant of mrc1, suggesting that Mrc1 regulates early origin firing in a checkpoint-independent manner (20). However, the mechanisms by which Mrc1 regulates early-firing origins are unknown.…”

The Amazing Acrobat: Yeast’s Histone H3K56 Juggles Several Important Roles While Maintaining Perfect Balance

“…They are thought to be a failsafe mechanism in case of fork collapse [66]. Cells deleted for Mrc1, which is found both at the checkpoint and at the replisome [67], showed replication from a greater number of dormant origins than the WT [68]. This over-abundance of firing leaves Mrc1 mutants with very few dormant origin reserves in case of fork stall and collapse.…”