Academic journal article Genetics

Fission Yeast Hsk1 (Cdc7) Kinase Is Required after Replication Initiation for Induced Mutagenesis and Proper Response to DNA Alkylation Damage

Academic journal article Genetics

Fission Yeast Hsk1 (Cdc7) Kinase Is Required after Replication Initiation for Induced Mutagenesis and Proper Response to DNA Alkylation Damage

Article excerpt

ABSTRACT

Genome stability in fission yeast requires the conserved S-phase kinase Hsk1 (Cdc7) and its partner Dfp1 (Dbf4). In addition to their established function in the initiation of DNA replication, we show that these proteins are important in maintaining genome integrity later in S phase and G2. hsk1 cells suffer increased rates of mitotic recombination and require recombination proteins for survival. Both hsk1 and dfp1 mutants are acutely sensitive to alkylation damage yet defective in induced mutagenesis. Hsk1 and Dfp1 are associated with the chromatin even after S phase, and normal response to MMS damage correlates with the maintenance of intact Dfp1 on chromatin. A screen for MMS-sensitive mutants identified a novel truncation allele, rad35 (dfp1-(1-519)), as well as alleles of other damage-associated genes. Although Hsk1-Dfp1 functions with the Swi1-Swi3 fork protection complex, it also acts independently of the FPC to promote DNA repair. We conclude that Hsk1-Dfp1 kinase functions post-initiation to maintain replication fork stability, an activity potentially mediated by the C terminus of Dfp1.

THE Hsk1 protein kinase, the fission yeast ortholog of Saccharomyces cerevisiae Cdc7, is a conserved protein essential for the initiation of DNA replication (Masai et al. 1995; Brown and Kelly 1998; Snaith et al. 2000). Data from many systems suggest that the kinase functions at individual replication origins to activate the prereplication complex (preRC) through phosphorylation of the MCM helicase and other subunits (reviewed in Forsburg 2004). In fission yeast, Hsk1 kinase activity is limited to S phase by its regulatory subunit Dfp1, which is transcriptionally and posttranslationally regulated to restrict its peak of activity to S phase (Brown and Kelly 1999; Takeda et al. 1999). The requirement for Dfp1 (in S. cerevisiae, Dbf4) is similar to the dependence of CDK kinases on cyclin activity; thus, the Ccd7 kinase family has been dubbed DDK (Dbf4-dependent kinases) ( Johnston et al. 1999; Duncker and Brown 2003). Hsk1 is a target of the Cds1 checkpoint kinase and undergoes Cds1-dependent phosphorylation during hydroxyurea (HU) treatment in vivo and in vitro (Snaith et al. 2000). Interestingly, deletion of Δcds1 partly rescues hsk1-1312 temperature sensitivity, which suggests that Hsk1 is negatively regulated by the replication checkpoint. In turn, Cds1 is poorly activated in hsk1 mutants after HU treatment, indicating that there may be a feedback loop linking these two kinases (Snaith et al. 2000; Takeda et al. 2001). hsk1 mutants are sensitive toHUtreatment, with a phenotype suggesting a specific defect in recovery (Snaith et al. 2000).

DDKkinases have substrates outside of the replication initiation pathway. Functional dissection of Schizosaccaromyces pombe Dfp1 identifies separate regions that are required for checkpoint response (N-terminal domain; Takeda et al. 1999; Fung et al. 2002), for centromere cohesion and replication (MIR domain; Bailis et al. 2003; Hayashi et al. 2009) and for proper response to alkylation damage during S phase (C-terminal domain; Takeda et al. 1999; Fung et al. 2002). Recent studies indicate that the DDK kinase is required for initiation of programmed double-strand breaks in meiosis (Sasanuma et al. 2008; Wan et al. 2008) and meiotic chromosome orientation (Lo et al. 2008; Matos et al. 2008). The different domains of Dfp1 are presumed to target the Hsk1 kinase to different substrates. Because kinase activity is limited to S phase, these results suggest that the cell uses the DDK kinase to link various cell-cycle events to S-phase passage.

MMScauses alkylation damage that affects replication forks (Wyatt and Pittman 2006; Kaina et al. 2007). This results in Cds1-dependent slowing of DNA replication forks (Lindsay et al. 1998; Marchetti et al. 2002). However, Δcds1 mutants are only modestly sensitive to MMS treatment (Lindsay et al. 1998;Marchetti et al. 2002), suggesting at least partial independence from the replication checkpoint. …

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