2634

Abstract

An unrepaired chromosomal double strand break (DSB) can initiate apoptosis while inhibition of apoptosis can lead to genomic instability. This suggests DNA repair and apoptotic pathways regulate one another; however, the mechanism of this inter-regulation is unknown. Prior studies have demonstrated that BRCA1 promotes DSB repair via homology-directed recombination (HDR) when nuclear and induces apoptosis when cytoplasmic. Likewise, proapoptotic Bid has been shown to regulate cell cycle checkpoints upon phosphorylation by ATM in response to DNA damage. We hypothesize that BRCA1 plays an important role in linking DNA repair and apoptosis through Bid. To examine this we asked 1) whether BRCA1 and Bid interact in response to DNA damage and 2) whether Bid affects HDR-mediated DSB repair. To test the interaction of BRCA1 and Bid in response to DNA damage, co- immunoprecipitations (co-IP) were performed in mouse neuronal HT-22 cells following 3 Gy ionizing radiation (IR) by IPing BRCA1 and immunoblotting for Bid. The reciprocal co-IP was also performed. To study the role Bid plays in HDR, we used a plasmid based chromosomal HDR assay. Chromosomal DSBs were induced with the expression of I-SceI. DSB-induced HDR resulted in restoration and expression of GFP and was quantified by FACS. To deplete Bid for the HDR assay, a siRNA approach was used. Our preliminary results show that BRCA1 and Bid exist in the same immunocomplex. This signal was enhanced 1 and 6 hours after 3 Gy IR. Knockdown of >90% of Bid was achieved with Bid-specific siRNA within 36 hours of transfection and remained depleted for 36 hours. Control siRNA had no effect on Bid level. Interestingly, knockdown of Bid resulted in a 2-fold reduction in HDR compared to control cells. The results from this study show that BRCA1 may interact with Bid, indicating potential functional inter-regulation between BRCA1 and Bid. Also, Bid plays a role in HDR-mediated chromosomal double strand break repair. We are in the process of (1) defining the molecular mechanisms of Bid regulating HDR and (2) determining the functional interplay of BRCA1 and Bid in the context of interconnecting DSB repair and apoptosis. Disconnection of these two processes may be essential in radiation-induced carcinogenesis and cancer cell resistance to radiotherapy.