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Vol. 19, Issue 12, 5550-5558, December 2008

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The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

Prasanta K. Patel^*, Naveen Kommajosyula^*, Adam Rosebrock, Aaron Bensimon^,, Janet Leatherwood, John Bechhoefer^||, and Nicholas Rhind^*

*Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605; Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, NY 11794; Genomes Stability Unit, Pasteur Institute, 75724 Paris, France; and ^||Department of Physics, Simon Fraser University, Burnaby, BC, Canada V5A 1S6

Submitted June 24, 2008; Revised August 28, 2008; Accepted September 8, 2008
Monitoring Editor: Daniel J. Lew

InCytes from MBC

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency.

Author contributions: P.K.P. collaborated with N. R. in designing experiments and analyzing data and performed the experiments in Figures 1, 2, and 4A and Supplemental Figure S1; N. K. performed the experiments in Supplemental Figures S2 and S3; A. R. and J. L. performed the experiment and analyzed the data in Supplemental Figure S4; A. B. provided the DNA combing surfaces; J. B. developed the mathematical tools for analyzing the FLIP data in Figure 3; and N. R. collaborated with P.K.P. in designing experiments and analyzing data, performed the experiments in Figures 3 and 4B, and wrote the paper.

Present address: Genomic Vision, Paris Santé Cochin, 29 rue du Faubourg Saint Jacques, 75014 Paris, France.

Address correspondence to: Nicholas Rhind (nick.rhind{at}umassmed.edu).

Abbreviations used: BrdU, 5-bromo-2'-deoxyuridine; DDK, Dbf4-dependent kinase; HU, hydroxyurea; ORC, origin recognition complex; pre-RC, pre-replication complex; YES, yeast extract plus supplements.

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