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

Abstract

To investigate the checkpoint response to aberrant initiation, we analyzed the cell cycle checkpoint response induced by mutations of Schizosaccharomyces pombe DNA primase. DNA primase has two subunits, Spp1 and Spp2 (S. pombe primases 1 and 2). Spp1 is the catalytic subunit that synthesizes the RNA primer, which is then extended by DNA polymerase ␣ (Pol␣) to synthesize an initiation DNA structure, and this catalytic function of Pol␣ is a prerequisite for generating the S-M phase checkpoint. Here we show that Spp2 is required for coupling the function of Spp1 to Pol␣. Thermosensitive mutations of spp2 ؉ destabilize the Pol␣-primase complex, resulting in an allele-specific S phase checkpoint defect. The mutant exhibiting a more severe checkpoint defect also has a higher extent of Pol␣-primase complex instability and deficiency in the hydroxyurea-induced Cds1-mediated intra-S phase checkpoint response. However, this mutant is able to activate the Cds1 response to S phase arrest induced by temperature. These findings suggest that the Cds1 response to the S-phase arrest signal(s) induced by a initiation mutant is different from that induced by hydroxyurea. Interestingly, a pol␣ts mutant with a defective S-M phase checkpoint and an spp2 mutant with an intact checkpoint have a similar Pol␣-primase complex stability, and the Cds1 response induced by hydroxyurea or by the mutant arrests at the restrictive temperature. Thus, the Cds1-mediated intra-S phase checkpoint response induced by hydroxyurea can also be distinguished from the S-M phase checkpoint response that requires the initiation DNA synthesis by Pol␣.

Role of Fission Yeast Primase Catalytic Subunit in the Replication Checkpoint

“…In vitro work suggests that activated Cds1 and Chk1 kinase can phosphorylate two regulators of the mitotic inducer Cdc2, Wee1, and Cdc25 (Furnari et al, 1997;Boddy et al, 1998;Zheng et al, 1998;Lopez-Girona et al, 1999). A number of replication mutants are synthetic lethal in cds1⌬ background but not in chk1⌬ background (Bhaumik and Wang, 1998;Tan and Wang, 2000). Furthermore, deletion of cds1 ϩ lowers the semipermissive temperature and exacerbates the mutation rate of several thermosensitive replication mutators (Liu et al, 1999).…”

“…Analysis of spp2 ϩ thermosensitive mutants has indicated that mutations of spp2 ϩ affect the stability of the Pol␣-primase complex. Maintaining a stable Pol␣-primase complex by Spp2 protein is required for the activation of the Cds1-mediated intra-S phase checkpoint (Tan and Wang, 2000).…”

“…We have analyzed the mutational effects of pol␣ ϩ and spp2 ϩ on the cell cycle checkpoint responses (Bhaumik and Wang, 1998;Tan and Wang, 2000). Mutational studies of fission yeast pol␣ ϩ indicate that cells with pol␣⌬ enter mitosis with 1C DNA content, and cells harboring a catalytically dead but physically intact Pol␣ in the Pol␣-primase complex enter mitosis inappropriately.…”

“…The catalytic subunit of primase, named p49 in mammalian cells and PRI1 in budding yeast, synthesizes the RNA primer. The second subunit, named p58 in mammalian cells, PRIII in budding yeast, and Spp2 in fission yeast, is required for coupling the RNA primer synthesis by primase catalytic subunit with Pol␣ for initiation DNA (iDNA) synthesis (Tan and Wang, 2000).…”

Replication stress activates DNA polymerase alpha-associated Chk1

“…p49 is the catalytic primase subunit that synthesizes the RNA primer, whilst the p58 subunit couples p49 to p180 and is thought to play a role in controlling RNA primer synthesis. 8,9 In Schizosaccharomyces pombe, temperature sensitive mutants of DNA Polα cause activation of Chk1. 10,11 In Xenopus, uncoupling of helicase and DNA polymerase activity during replication results in Polα-dependent Chk1 activation.…”