Dissection of the cellular response to reduced DNA replication initiation events following inhibition of HSK1-DFP1 in Schizosaccharomyces pombe with chemical genetics

In eukaryotic cells, initiating replication during S phase requires the activity of the Dbf4-Dependent Kinase (DDK), which is composed of the heterodimer Hskl-Dfpl in Schizosaccharonzyces pornbe. To investigate the cellular response to a reduction in replication initiation events, a novel allele of hskl (hsklAS) was generated for use in a chemical genetics approach to conditionally inactivate the kinase, Hskl AS, in vivo using the small molecular inhibitor 3MB-PP I. Inhibition of Hsk1AS results in a rapid reduction in Mcm2 phosphorylation as well as a delay in S phase progression not observed in uninhibited cells. Consistent with a delay in S phase progression, inhibition of HsklAS prior to S phase entry decreases the frequency of initiation events, measured by observing replication intermediates on single DNA fibers. Inhibition of HsklAS prior to S phase with varying concentrations of 3MB-PPl also delays the subsequent mitosis in a dose dependent manner. The delay in mitotic entry is dependent on components of the DNA damage checkpoint, Rad3 and Chkl, which are also required to maintain cell viability following HsklAS inhibition. Following release from hydroxyurea arrest Hsk1AS inhibition does not affect DNA chain elongation, although entry into the subsequent mitosis is again delayed as evidenced by a delay in the separation of spindle pole bodies relative to uninhibited cells. The addition of caffeine abrogates much of this observed delay. The replication checkpoint is not required to delay mitosis in hsklAS cells released from hydroxyurea in the presence of 3MB-PP1, although Chkl is necessary to prevent irreversible chromosome segregation defects. Together, these results suggest that a reduction in initiation events during S phase generates DNA structures that are recognized by the cell. A reliance on Rad3 and Chkl to maintain viability and protect genomic integrity following Hskl AS inhibition suggests that this cellular recognition occurs via checkpoint activation and may represent a primary reason for the evolution of Chkl. These results may provide insight into a protective mechanism employed by normal cells not present in a number of cancer cells and further understanding of this mechanism could aid in the development of additional anticancer therapeutics.