Program Project in Structural Cell Biology of DNA Repair Machines: Project 4: Homologous Recombination Repair (HRR)

Performance Sites

Biology and Biotechnology Research Program
Lawrence Livermore National Laboratory 
7000 East Avenue
P.O. Box 808, L441
Livermore, CA 94551

Univ. of California at Davis
Department of Microbiology and Molecular & Cellular Biology
Hutchison Hall Room 258
Davis, CA 95616-8665

UC / Lawrence Berkeley National Laboratory
1 Cyclotron Road
Life Sciences Division, Building 74
Berkeley, CA 94720

Key Personnel

Name	Organization	Role on Project
Thompson, Larry	Lawrence Livermore National Laboratory	Project Leader
Kowalczykowski, Stephen	Univ. of California at Davis	Senior Investigator
Schild, David	UC / Lawrence Berkeley National Laboratory	Senior Investigator
Mazin, Alexander	Univ. of California at Davis	Research Scientist
Heyer, Wolf-Dietrich	University of California at Davis	Collaborator
Lee, Wen-Hwa	U. Texas Health Science Center at San Antonio	Collaborator
Liu, Nan	Lawrence Livermore National Laboratory	Collaborator
Sung, Patrick	U. Texas Health Science Center at San Antonio	Collaborator
Takata, Minoru	Kawasaki Medical School, Kurashiki, Japan	Collaborator
Takeda, Shunichi	Faculty of Medicine, Kyoto University, Japan	Collaborator

Links to web sites:

SBDR Project 4 Abstract

In human cells, as in Saccharomyces cerevisiae, homologous recombinational repair (HRR) is a major 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 DNA strand-transferase and other proteins that include distant relatives of Rad51, which are referred to as 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 yeast 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 mediating the BRCA2 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, reconstitution studies using purified yeast and human HRR proteins in established assays will also address the molecular mechanism(s) by which Rad51 paralogs ensure chromosome stability and prevent tumorigenesis.

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