Defense Date


Document Type


Degree Name

Doctor of Philosophy



First Advisor

Kristoffer Valerie

Second Advisor

Suzanne Barbour

Third Advisor

Lawrence Povirk

Fourth Advisor

Sumitra Deb

Fifth Advisor

Swati Deb


Mutations in the breast cancer susceptibility 1 (BRCA1) gene are linked to breast as well as ovarian cancers. However, most cancer-causing mutations within the BRCA1 gene have been found in the N’ and C’ terminal regions of the BRCA1 protein, both believed to be important for DNA double-strand break (DSB) repair. The BRCA1 C’ terminal (BRCT) repeats have been implicated in phospho-serine protein binding whereas the N’ terminal RING domain interacts with the BARD1 protein to form a hetero-dimeric complex with E3 ubiquitin ligase activity. The BRCA1 BRCT domain binds CtIP, BACH1, and RAP80, all of which have been directly implicated in homologous recombination repair (HRR). Lysine 1702 (K1702) of BRCA1 resides within the phospho-serine binding pocket of the first BRCT repeat of BRCA1. To determine the effect of manipulating the ability of BRCA1 to bind CtIP and other phospho-proteins binding to the BRCA1 BRCT domain on DSB repair, and specifically HRR, we introduced a K1702M mutation into BRCA1 known to impair BRCT binding to a pSer-X-X-Phe peptide representing BACH1. Surprisingly, instead of impairing HRR, we found that BRCA1 K1702M resulted in hyper-recombination with > 3-fold higher levels of HRR compared to wild-type BRCA1 using an HRR assay based on GFP expression in BRCA1-defective HCC1937 cells. This hyper-recombinogenic phenotype coincided with cell-cycle arrest in S/G2 suggesting that the potential lack of binding of critical proteins to the BRCA1 BRCT domain results in abnormal HRR by priming cells to undergo more HRR which is enhanced during the S and G2 phases of the cell-cycle. In line with the increased HRR seen with the HRR/GFP assay, HCC1937 cells expressing BRCA1 K1702M showed increased levels of RAD51 foci and nuclear staining suggesting that HRR was highly elevated. Interestingly, the hyper-recombinogenic phenotype of BRCA1 K1702M could be reduced to normal levels with a second mutation (I26A) in BRCA1 that affects BRCA1 and CtIP ubiquitination. These results reveal a hierarchal regulation of HRR with ubiquitination having a dominate role in DSB repair by BRCA1 and suggests that targeted disruption of BRCT-CtIP binding increases HRR that is in turn controlled by ubiquitination. In addition, we provide evidence that BRCA1 serine 1387 phosphorylation within the SQ cluster region of BRCA1 is involved in the cell survival and DNA damage response to IR. The BRCA1 S1387A mutant only partially increased the radiosurvival of HCC1937 cells compared to cells expressing wild-type BRCA1 and immunocytochemical analysis revealed wild-type BRCA1 was located in the nucleus whereas the S1387A mutant was cytoplasmic in response to IR. We also show that BRCA1 SQ cluster serine phosphorylation in addition to serine 1387 is involved in HRR. Altogether, these findings reveal the importance of various regions of BRCA1 in DSB repair and may lead to multiple strategies of modulating BRCA1 function in response to DNA damage.


© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

May 2009