The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

• DOI: 10.1091/mbc.e08-06-0645
• Publication Date: 2008-09-18T00:53:59Z
• Authors: Prasanta K. Patel; Naveen Kommajosyula; Adam Rosebrock; Aaron Bensimon; Janet Leatherwood; John Bechhoefer; Nicholas Rhind
• Keywords: Cell Nucleus; DNA Replication; 0301 basic medicine; 570; 0303 health sciences; 500; Cell Cycle Proteins; Replication Origin; Protein Serine-Threonine Kinases; 03 medical and health sciences; Chromosomal Instability; Gene Expression Regulation, Fungal; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Protein Kinases; Fluorescence Recovery After Photobleaching; Plasmids

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.