Role of fission yeast primase catalytic subunit in the replication checkpoint

To investigate the cell cycle checkpoint response to aberrant S phase-initi ation, we analyzed mutations of the two DNA primase subunit genes of Schizo saccharomyces pombe, spp1(+) and spp2(+) (S. pombe primase 1 and 2). spp1() encodes the catalytic subunit that synthesizes the RNA primer, which is t hen utilized by Pol alpha to synthesize the initiation DNA. Here, we report ed the isolation of the fission yeast spp1(+) gene and cDNA and the charact erization of Spp1 protein and its cellular localization during the cell cyc le. Spp1 is essential for cell viability, and thermosensitive mutants of sp p1(+) exhibit an allele-specific abnormal mitotic phenotype. Mutations of s pp1(+) reduce the steady-state cellular levels of Spp1 protein and compromi sed the formation of Pol alpha -primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 che ckpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Pol alpha has previously shown that activation of the replication checkpoin t requires the initiation of DNA synthesis by Pol alpha. Together, these ha ve led us to propose that suboptimal cellular levels of pol alpha -primase complex due to the allele-specific mutations of Spp1 might not allow Pol al pha to synthesize initiation DNA efficiently, resulting in failure to activ ate a checkpoint response. Thus, a functional Spp1 is required for the Chk1 -mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.