We have identified thermosensitive mutants of five Schizosaccharomyces pombe replication proteins that have a mutator phenotype at their semipermissive temperatures. Allele-specific mutants of DNA polymerase ␦ (pol␦) and mutants of Pol␣, two Pol␦ subunits, and ligase exhibited increased rates of deletion of sequences flanked by short direct repeats. Deletion of rad2 ؉ , which encodes a nuclease involved in processing Okazaki fragments, caused an increased rate of duplication of sequences flanked by short direct repeats. The deletion mutation rates of all the thermosensitive replication mutators decreased in a rad2⌬ background, suggesting that deletion formation requires Rad2 function. The duplication mutation rate of rad2⌬ was also reduced in a thermosensitive polymerase background, but not in a ligase mutator background, which suggests that formation of duplication mutations requires normal DNA polymerization. Thus, although the deletion and duplication mutator phenotypes are distinct, their mutational mechanisms are interdependent. The deletion and duplication replication mutators all exhibited decreased viability in combination with deletion of a checkpoint Rad protein, Rad26. Interestingly, deletion of Cds1, a protein kinase functioning in a checkpoint Rad-mediated reversible S-phase arrest pathway, decreased the viability and exacerbated the mutation rate only in the thermosensitive deletion replication mutators but had no effect on rad2⌬. These findings suggest that aberrant replication caused by allele-specific mutations of these replication proteins can accumulate potentially mutagenic DNA structures. The checkpoint Rad-mediated pathways monitor and signal the aberrant replication in both the deletion and duplication mutators, while Cds1 mediates recovery from aberrant replication and prevents formation of deletion mutations specifically in the thermosensitive deletion replication mutators.Acquired genetic instability has been proposed to be an early event in tumor evolution (24,25). The hypothesis that cancer cells exhibit a mutator phenotype or an increased rate of mutation has been validated by the finding that mutations in mismatch repair (MMR) genes lead to microsatellite instability and are the underlying cause of both hereditary nonpolyposis colon cancer (HNPCC) and sporadic tumors (reviewed in references 18, 23, and 26). In addition to MMR genes, mutations in other genes responsible for ensuring genomic stability may also generate a mutator phenotype (25). Thus, proteins that maintain genomic stability in normal cells, including those involved in DNA replication, repair, recombination, chromosomal segregation, transcription, and cell cycle control are prime proto-mutator candidates. Mutations in any of these genes may be an early event in tumorigenesis, allowing the generation of the multiple mutations observed in cancers.In addition to microsatellite instability, another common source of mutation in human genetic diseases is deletion of sequences flanked by short direct repeats (19). For example...

In hematopoietic cells, g-irradiation causes a p53-dependent transient G1 phase cell cycle arrest. Various extracellular growth inhibitory signals elicit G1 arrest by targeting CDK4. Here we show that in a myeloid cell line, 32D cl 3, enforced expression of CDK4, but not cyclins D2 nor D3, overrides the g-irradiation-induced G1 arrest. CDK4 does not confer resistance to the radiation-induced G2 block observed in parental cells. Ectopic expression of CDK4 overcomes the ionizing radiation-induced inhibition of CDK4 and CDK2 kinase activity. The levels of CDK4 protein do not change after exposure to ionizing radiation in either parental cells or those overexpressing CDK4. Ionizing radiation induces the expression of both p53 and p21, and in cells constitutively synthesizing exogenous CDK4, the return of p53 protein levels to baseline is prolonged. Increased levels of p21 are found associated with CDK4, and not CDK2, in the lines overexpressing CDK4, compared to the parental line, after exposure to ionizing radiation. Enforced expression of CDK4 may therefore overcome a g-irradiation-induced G1 arrest through the titration of the CDK inhibitor p21 allowing both CDK4 and CDK2 to remain active.

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 ϩ and spp2 ϩ (S. pombe primase 1 and 2). spp1 ϩ encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Pol␣ to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1 ϩ 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 ϩ exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1 ϩ 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.