Regulation of replication timing in fission yeast
Abstract
Here we report the first characterization of replication timing and its regulation in the fission 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 specific 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.
Acknowledgements
This publication would not have been possible without important contributions from Amy Piwowar, who collected and analyzed the quantitative fluorescence microscopy data (Figure 1), from Marius Poitelea, who assisted in development of the cdc10 synchronization protocol, from Deborah Mahoney, who initiated replication timing experiments in our laboratory and from Dharani Dubey, who helped characterize ars727, ars745 and ars766, and provided useful criticism of the manuscript. We are grateful to Michelle Calos, Louise Clarke, Elaine Nimmo, Robin Allshire and Francisco Antequera for plasmids. We thank Matthew O’Connell and Tony Carr for the cdc10-M17 strain, Tony Carr for the rad3Δ strain and Hiroshi Murakami and Hiroto Okayama for the cds1Δ strain. This research was supported by grants to J.A.H. from the National Institutes of Health (GM49294 and CA84302).