The Functions of BRCA2 in Homologous Recombinational Repair
Annual rept. 1 Jul 2003-30 Jun 2004
CALIFORNIA UNIV BERKELEY
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Using an HR assay system, we found that individual expression of several small BRCA2 regions in human HT1080 cells causes a reduced frequency in homologous recombination. Our provide the direct cellular evidence that the BRCA2-Rad51 interaction is crucial for HR repair and multiple regions of BRCA2 pretein are involved in regulating HR repair. Using the baculovirus co expression and Ni-NTA pull-down strategies, we demonstrated that BRCA2 forms a multiprotein complex with Rad51, Rad51B and Rad51C DNA repair proteins involving a strong interaction between BRCA2 and Rad51, and between Rad51B and Rad51C. A weak interaction between Rad51B and Rad51C was observed as well. We also found that the BRC repeats of BRCA2 do not directly interact with Rad51B or Rad51C. In addition, we have successfully expressed three BRC fragments using baculovirus expression system. These protein expressions were confirmed by Western analysis. The purification of these proteins was found to be difficult because these proteins were extremely unstable and tended to be degraded during the purification process. We have tested several conditions to stabilize the proteins, including use of different salts, different concentration of salts, different expression temperature, and co-expression of the proteins with Rad51. We have established three biochemical assays for Rad51 activities, including DNA binding, ATPase and DNA strand exchange. The investigation regarding whether the BRC1-4, BRC5-8 or BRC1-8 proteins affects the Rad51 activities is underway.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research