Our ultimate goal is to understand the molecular mechanisms of chromosome segregation. Specifically, we want to understand how chromosomes are replicated precisely, how their structural integrity is maintained, and how their separation is coordinated with other events in the cell cycle. To probe these issues, we will study mutations in two genes that encode biochemically well-characterized proteins that may play essential roles in both the mechanics of DNA replication and its coordination with mitosis. CDC44 (the large subunit of replication factor C) and PCNA (proliferating cell nuclear antigen) have been shown to interact both biochemically and genetically, and each has characteristics that suggest a dual role in the cell cycle. Thorough biochemical analysis reveals that replication factor C and PCNA act together to promote the processivity of DNA polymerase delta in vitro. Nonetheless, cell-cycle analysis of a cdc44 mutant suggests that a critical mitotic (post-replicative) function is perturbed. Correspondingly, the association of PCNA with most cyclin-dependent-kinase complexes in vivo suggests that PCNA may play an important role in the coordination of DNA synthesis with other cellular events. To analyze the roles of CDC44 and PCNA in living cells we will create random and site-directed mutations in vitro and analyze the phenotypes they confer. Perturbations of DNA replication will be signaled by elevated mutation rates, disrupted DNA synthesis, and an appropriate cell-cycle arrest. Perturbations of mitotic functions will be revealed by a post- replicative arrest or phenotypes characteristic of a regulatory defect. Because CDC44 and PCNA appear to be components of a large intracellular complex, complete understanding of their roles in the cell will require an understanding of the components with which they interact. Thus, pseudoreversion and unlinked non-complementation will be used to identify functional interactions that take place in vivo. Mutagenesis and phenotypic analysis will then be used to examine the roles of the newly identified gene products in the vital processes of the cell cycle. A related project will examine the effect on telomere metabolism of perturbing DNA replication. The allele-specific elongation of telomeres in cdc44 and cdcl7 (DNA polymerase delta) mutants suggests that specific perturbations of DNA replication can unbalance the processes that maintain telomeres. To determine the characteristics that lead to this imbalance, telomere elongation will be assessed in cdc44 and cdcl7 mutants with different phenotypes, and the effects of cell-cycle delays will be examined. Taken together, our studies of CDC44 and PCNA will lead to an improved understanding of DNA replication and the cell cycle, and ultimately of genetic diseases such as cancer.
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