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Vol. 12, Issue 1, 115-128, January 2001
and
*Department of Pathology, Stanford University School of Medicine,
Stanford, California 94305-5324; and To investigate the cell cycle checkpoint response to aberrant S
phase-initiation, we analyzed mutations of the two DNA primase subunit
genes of Schizosaccharomyces pombe,
spp1+ and spp2+
(S. pombe primase 1 and 2).
spp1+ encodes the catalytic subunit that
synthesizes the RNA primer, which is then utilized by PolImperial Cancer
Research Fund, London WC2A 3PX, United Kingdom
to
synthesize the initiation DNA. Here, we reported the isolation of the
fission yeast spp1+ gene and cDNA and the
characterization of Spp1 protein and its cellular localization during
the cell cycle. Spp1 is essential for cell viability, and
thermosensitive mutants of spp1+ exhibit an
allele-specific abnormal mitotic phenotype. Mutations of
spp1+ reduce the steady-state cellular
levels of Spp1 protein and compromised the formation of Pol-primase
complex. The spp1 mutant displaying an aberrant mitotic
phenotype also fails to properly activate the Chk1 checkpoint kinase,
but not the Cds1 checkpoint kinase. Mutational analysis of Pol has
previously shown that activation of the replication checkpoint requires
the initiation of DNA synthesis by Pol. Together, these have led us
to propose that suboptimal cellular levels of pol-primase complex
due to the allele-specific mutations of Spp1 might not allow Pol to
synthesize initiation DNA efficiently, resulting in failure to activate
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.
Corresponding author. E-mail
address: twang{at}cmgm.stanford.edu.
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