This proposal addresses a fundamental question in cell biology: how do eukaryotic cells execute the processes of DNA replication and DNA repair? The relevance of this research to human health is underscored by the increasing number of cancers that have been linked to mutations in genes that are involved in these processes. Previously, I have characterized the phenotypes of yeast cells bearing mutations in the genes that encode subunits of RFC and PCNA. RFC and PCNA are two essential DNA polymerase accessory proteins that are required both for DNA replication and for the repair of DNA damage caused by alkylating agents and Uv irradiation. I will use a combined biochemical and genetic approach to investigate how these proteins function in DNA replication and repair, and I will study how the completion of these processes is linked with the regulatory controls that coordinate cell cycle progression. First, I will undertake a biochemical structure/function analysis of wild type and mutant RFC and PCNA proteins in vitro. This analysis will include assays of DNA-binding, nucleotidebinding, and RFC-PCNA-DNA complex formation. In order to identify gene products that interact with RFC and PCNA, I will isolate and characterize mutant alleles of genes that exhibit synthetic lethality with mutant RFC and PCNA alleles. Additionally, I will use second-site suppressor analysis to identify gene products that alleviate the UV irradiation-sensitive phenotype of RFC mutants. I will also investigate how the presence of DNA damage delays cell cycle transitions. I will characterize the UV-induced DNA damage signal that triggers the G1/S phase checkpoint, and I will test whether the Cdc28p cyclin dependent kinase is a target of this checkpoint mechanism. I will also use a genetic screen to identify gene products that mediate the cdc44-induced cell cycle arrest. These experiments will provide detailed information of how the processes of DNA replication and DNA repair are executed in S. cerevisiae.
| Ionescu, Costin N; Shea, Kathleen A; Mehra, Rajendra et al. (2002) Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC. Biochemistry 41:12975-85 |
| Beckwith, W; McAlear, M A (2000) Allele-specific interactions between the yeast RFC1 and RFC5 genes suggest a basis for RFC subunit-subunit interactions. Mol Gen Genet 264:378-91 |
| Beckwith, W H; Sun, Q; Bosso, R et al. (1998) Destabilized PCNA trimers suppress defective Rfc1 proteins in vivo and in vitro. Biochemistry 37:3711-22 |
