Regulation of replication timing in fission yeast

Abstract

Here we report the ®rst characterization of replication timing and its regulation in the ®ssion yeast Schizosaccharomyces pombe. We used three different synchronization methods: centrifugal elutriation, cdc10 temperature-shift and release, and starvation for deoxyribonucleoside triphosphates (dNTPs) by treatment with hydroxyurea (HU) followed by removal of HU, to study the times when speci®c autonomously replicating sequence elements (ARS elements; potential replication origins) replicate during S phase. We found that individual ARS elements replicate at characteristic times, some early and some late, independently of synchronization method. In wild-type cells treated with HU, early ARS elements replicated but late ones did not. However, in HU-treated mutant cells lacking the Rad3 (similar to human ATR and ATM) or Cds1 (similar to human CHK2) checkpoint kinase, both early and late ARS elements were able to replicate. Thus under conditions of dNTP starvation the Rad3 and Cds1 kinases are needed to suppress the replication of normally late-replicating regions.

Stochastic hybrid modeling of DNA replication across a complete genome

“…As expected, however, the predicted average number of firing origins also increases from 18% to 57%. While small values of T f (high firing propensities) lead to reasonable S-phase completion times, the high number of firing origins predicted for these values is not supported by experimental data (22,24,27,31).…”

Regulation of Cellular and SV40 Virus Origins of Replication by Chk1-dependent Intrinsic and UVC Radiation-induced Checkpoints

“…In fact, mutations in the budding yeast checkpoint genes RAD53 and ORC2 similarly abrogate both types of checkpoints, one that regulates late S origins in cells blocked in early S phase that, in checkpoint defective strains, are activated in these cells with normal kinetics (16,18,36), and an intrinsic checkpoint that targets only some late S origins, which are activated in an accelerated fashion in checkpoint defective strains in the absence of a replication block or other perturbations (16). Furthermore, mutations in Rad3, the fission yeast homologue of ATR, abrogate both a replication arrest-dependent checkpoint that targets late S origins (19) and an intrinsic checkpoint that regulates some, but not all of these origins in the absence of a replication block. 2 The existence of a Chk1 and ATR-dependent intrinsic checkpoint is consistent with the observation in Xenopus of an increased association of ATR with chromatin during DNA replication unperturbed by drugs (46).…”

“…In budding yeast, this and a similar checkpoint that responds to stalled replication forks repress late S phase origins of replication in early S phase cells (16 -18). In both fission (19) and budding (17) yeast, this latter checkpoint also requires homologues of ATR and ATM, in addition to other checkpoint proteins. A similar checkpoint in mammals requires the checkpoint kinase Chk1 (20) and is abrogated by the ATM and ATR inhibitor caffeine (21).…”

DNA Replication Origins Fire Stochastically in Fission Yeast

“…In the one case in which origin efficiency has been directly quantitated, ORI19 fires in 40% of S phases and ORI22 fires in 30% (Segurado et al, 2002). In other examples, one of the most efficient fission yeast origins, ars2-1, appears to fire in no more than 50% of S phases; others appear to fire in Ͻ30% of S phases (Dubey et al, 1994;Gomez and Antequera, 1999;Kim and Huberman, 2001). Two very different examples of how origin firing can be organized are provided by budding yeast, in which firing is relatively well organized (Raghuraman et al, 2001;Yabuki et al, 2002) and frog embryos extracts, in which it is random (Herrick et al, 2000;Lucas et al, 2000;Blow et al, 2001;Marheineke and Hyrien, 2001;Herrick et al, 2002).…”