Analysis of Fission Yeast Primase Defines the Checkpoint Responses to Aberrant S Phase Initiation

To maintain genomic integrity, eukaryotic cells have the checkpoint mechanisms to delay progression of the cell cycle when cells encounter perturbation of DNA replication or DNA damage (18). In fission yeast, a group of proteins, Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1, known as checkpoint Rad proteins, function early in the surveillance of both the replication perturbation and DNA damage (1, 13). These checkpoint Rad proteins are thought to sense and transduce signals of aberrant replication and DNA damage to activate two downstream protein kinases, Cds1 and Chk1, to arrest the cell cycle (5–8, 40). In response to S phase arrest by hydroxyurea, cdc mutant arrest, or DNA damage induced during S phase, Cds1 is phosphorylated and activated (19). Cds1 activation delays the progression of S phase (termed intra-S phase checkpoint) and contributes to preventing mitosis (3, 19, 26). The Cds1 structural counterpart of budding yeast, RAD53, has been shown to be an essential factor for maintaining the intra-S phase checkpoint to prevent the firing of late replication origins when the progression of replication forks from early-firing origins is blocked by hydroxyurea (12, 33, 34). Another downstream kinase, Chk1, is required to arrest mitosis when DNA is damaged in late S phase and G2 phase and is also required to prevent mitosis when cdc replication mutants are used to perturb S phase (7). Chk1 is not required to prevent mitosis in response to hydroxyurea block. Following DNA damage, Chk1 protein is phosphorylated in a cell-cycle-specific manner (23), and Chk1 phosphorylation is correlated to cell cycle arrest (4). Chk1 phosphorylation allows binding of 14-3-3 proteins with Chk1 that is thought to direct Chk1 for specific substrate (9). Although Chk1 is not phosphorylated in hydroxyurea block or during early S phase perturbation (23), Chk1 is phosphorylated when S phase is blocked by hydroxyurea in a cds1Δ background (4, 19). Chk1 kinase has been shown to phosphorylate in vitro two Cdc2 kinase regulators, Wee1 kinase and Cdc25 phosphatase (15, 30, 32). Phosphorylation of Cdc25 by Chk1 allows Cdc25 to associate with 14-3-3 proteins, leading to nuclear exclusion of Cdc25 (21, 31, 42). These findings strongly suggest that checkpoint signals generated from early-S-phase perturbation are different from those generated during ongoing or late S phase. Early-S-phase perturbation activates Cds1 kinase to maintain an intra-S phase checkpoint, while ongoing or late-S-phase perturbation results in Chk1 phosphorylation to prevent mitosis. Thus, Cds1 and Chk1 function in two distinct but mutually reinforced ways in the cell cycle surveillance mechanisms. We are interested in defining the requirements for generating the checkpoint response to aberrant S phase initiation. To achieve this goal, we investigated the effect of mutations of DNA polymerase α (Polα)-primase on the cell cycle events. DNA Polα-primase, a four-subunit enzyme complex, is the principal enzyme that initiates DNA replication on both leading and lagging strands. DNA primase synthesizes an RNA primer which is then extended by Polα to synthesize an initiation DNA structure (39, 41). DNA primase is a heterodimeric enzyme complex, consisting of a catalytic subunit that synthesizes the RNA primer, named p49 in mammalian cells and PRI1 in budding yeast, and a second subunit that has no detectable enzymatic activity, named p58 in mammalian cells and PRIII in budding yeast (41). We and others have previously demonstrated that deletion or mutation of DNA Polα results in the cells entering inappropriate mitosis (2, 11). We have previously shown that germinating spores derived from a Schizosaccharomyces pombe heterozygous diploid containing one copy of the polα+ gene with a mutation at a critical metal activator binding residue (Asp984 in region I) display an abnormal mitotic phenotype (see Fig. 2 of reference 2). In vitro studies have shown that Polα-primase complex containing this specific Polα mutation abolishes only the catalytic function and not the physical association of Polα and primase, and the mutant complex is able to synthesize the RNA primer in vitro but is unable to extend the RNA primer (10). These experiments strongly suggest that the catalytic function of Polα is required for generating the checkpoint to prevent inappropriate mitotic entry. We isolated the fission yeast genes and cDNAs of both primase subunits, named spp1+ and spp2+ for S. pombe primases 1 and 2, respectively, and generated conditional mutants of spp1+ and spp2+. In this report, we investigate the effect of spp2 mutations on the cell cycle checkpoint response. The results of our studies indicate that Spp2 is the subunit that couples the function of Spp1 with Polα. Mutations of spp2 cause instability of Polα-primase complex. Analyses of the checkpoint effector kinase response to mutations of spp2 indicate that the requirement for Cds1 checkpoint response to an S phase initiation mutant arrest is different from that for Cds1 response to replication stall induced by the DNA synthesis inhibitor hydroxyurea. The requirements for intra-S phase Cds1 response can also be distinguished from the Polα activity-dependent S-M phase checkpoint response.