In human cells, as in Saccharomyces cerevisiae, homologous recombinatorial repair (HRR) is a major pathway that preserves chromosome integrity by removing double-strand breaks, crosslinks, and other DNA pathway that preserves chromosome integrity by removing double-strand breaks, crosslinks, and other DNA damage. HRR is critical for the error-free repair of double-strand breaks arising during normal DNA replication or exposure to ionizing radiation (IR). In yeast and human cells, this process is mediated by the highly conserved Rad51 paralogs. Mutations in both the mammalian and chicken Rad51 paralogs (XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D) cause excessive spontaneous chromosomal aberrations and sensitivity to IR and DNA crosslinks. The major goals of this project are to determine exactly how these Rad51 paralogs participate in HRR and how this pathway is regulated. This component of the Structural Cell Biology of DNA Repair Machines (SBDR) Program Project will employ structural and functional analyses of Biology of DNA Repair Machines (SBDR) Program Project will employ structural and functional analyses of yeast and human Rad51 (hRad512) and Rad51-paralog proteins, as well two hRad51 interactors the human and human Rad51 (hRad51) and Rad51-paralog proteins, as well two hRad51 interactors the human BRCA2 breast cancer onco-protein and the XPG protein. This dual approach will establish interfaces, define stable complexes, determine structures of dove-tailed domain complexes, and aid elucidation of the biological significance of defined interfaces via designed mutational analyses. Two Cores are essential for the success of this part of the SBDR Program Project. The Expression/Molecular Biology Core will enable efficient evaluation of multiple target-protein expression constructs and their optimization for crystallizations and structural investigations in the Structural Cell Biology Core. Since all Rad51 paralogs are candidate ATPases, potential ATP-driven conformational changes will be assessed by small angle x-ray scattering of proteins in solution (SAXS), and the role of ATP binding/hydrolysis in protein interactions and biological function will be defined. The residues of hRad51 media5ting the BRCA2 interaction will be defined by studying interaction will be defined by studying interaction-defective hRad51 mutations to reveal whether HRR depends on this interaction. The significance of a novel interaction of hRad51 with XPG will also be assessed. Finally, reconstitutions studies using purified yeast and human HRR proteins in established assays will also address the molecular mechanism(s) buy which Rad51 paralogs ensure chromosome stability and prevent tumorigenesis.
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