| Homologous recombination (HR) is involved in the creation of genetic diversity, maintenance of chromosome structure, and restoration of DNA damage. In mammals, efficient repair of double-strand breaks and interstrand crosslinks requires activities of Rad51 (a RecA homolog) and the Rad51-related proteins: Rad51B (Rad51L1), Rad51C (Rad51L2), Rad51D (Rad51L3), Xrcc2, Xrcc3, and Dmc1. RAD51 family genes demonstrate alternative splicing, and approximately 45% amino acid similarity is shared among their full-length translation products. All Rad51 proteins contain Walker box motifs consistent with an ability to bind and hydrolyze ATP. Two distinct complexes, Rad51B-Rad51C-Rad51D-Xrcc2 and Rad51C-Xrcc3, participate in early and late HR. With exception of Dmc1, deletions of the Rad51 genes in mouse are lethal; however, failure of Rad51d-null cells to proliferate can be overcome by disruption of the Trp53 tumor suppressor gene.; Initial studies described in this dissertation ascertained that Rad51d-deficient cells display reduced RAD51 foci following irradiation and hypersensitivity to various DNA damaging agents. Rescue of this phenotype by recombinant Rad51d cDNA expression establishes the involvement of RAD51D in homologous recombination repair. Manuscript 1 includes an analysis of Rad51d Walker box mutants demonstrating a respective 96% (K113R) and 83% (K113A) decrease in their ability to resist mitomycin C induced interstrand crosslinks. Yeast two-hybrid studies of these mutants display reduced ability (8-fold) to associate with RAD51C while interaction with XRCC2 is retained. These studies determine the ATP binding site in RAD51D is required for HR repair. Analysis of Rad51d alternative splice variants, presented in Manuscript 2, reveals each lack the ability to repair DNA interstrand crosslinks. Yeast two-hybrid experiments demonstrate the ALT2 variant binds both RAD51C and XRCC2 while ALT4/5 and ALT7 interacts specifically with either RAD51C or XRCC2 respectively. Domain mapping of splice variants identified portions of RAD51D that govern its partnership with RAD51-related proteins. These novel findings suggest RAD51D alternative isoforms potentially regulate HR. Finally, ongoing proteomics research has identified 72 RAD51D interacting candidates that include: MCM5, Histone H2A.1, Numatrin, Nucleotide binding protein 2, and PML. Continued investigations will determine how these interactions influence homologous recombination and the maintenance of chromosome stability. |
