The Functions of BRCA2 in Homologous Recombinational Repair
Annual rept. 1 Jul 2005-30 Jun 2006
TEXAS UNIV AT DALLAS SOUTHWESTERN MEDICAL CENTER
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We demonstrated that the BRCA2-Rad51 interaction is crucial for HR repair and multiple regions of BRCA2 protein are involved in regulating HR repair. Using biochemical approaches, we found 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. We successfully expressed three BRC fragments using baculovirus expression system. However, 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. The investigation regarding whether the BRC1-4, BRC5-8 or BRC1-8 proteins affects the Rad51 activities are underway. We found that, upon replication stresses, DNA-PKcs is phosphorylated and phosphorylated DNA-PKcs co-localizes with Brca1. The possible interaction between DNA-PKcs and Brca1 was confirmed in the co-immunoprecipitation co-IP analysis showing that DNA-PKcs could be co-precipitated with the -Brca1 antibody. Furthermore, GST-Brca1 fusion proteins covering different region of Brca1 were mixed with HeLa nuclear extract followed by co-IP with -DNA-PKcs antibody and western against -GST antibody. The result showed that Brca1 fragment 5 may potentially interact directly with DNA-PKcs.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research