RESEARCH PAPER


Mutations in the BRCT binding site of BRCA1 result in hyper-recombination

Seth M. Dever1,2, Sarah E. Golding1, Elizabeth Rosenberg1, Bret R. Adams1,2, Michael O. Idowu3, John M. Quillin4,6, Nicholas Valerie1, Bo Xu7, Lawrence F. Povirk1,5, and Kristoffer Valerie1,2,6
1Department of Radiation Oncology, Virginia Commonwealth University, Richmond, VA 23298, USA
2 Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA 23298, USA
3 Department of Pathology, Virginia Commonwealth University, Richmond, VA 23298, USA
4 Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA
5 Department Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298, USA
6 Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA;
7Department of Radiation Oncology, The Methodist Hospital, Houston, TX 77030, USA
Key words:
DNA repair, DNA damage, ubiquitination, radiation, PML bodies, breast cancer
Received:
427/11; Accepted: 5/5/11; Published: 5/8/11
Corresponding author:
E-mail:

Abstract

We introduced a K1702M mutation in the BRCA1 BRCT domain known to prevent the binding of proteins harboring pS-X-X-F motifs such as Abraxas-RAP80, BRIP1, and CtIP. Surprisingly, rather than impairing homologous recombination repair (HRR), expression of K1702M resulted in hyper-recombination coinciding with an accumulation of cells in S-G2 and no effect on nonhomologous end-joining. These cells also showed increased RAD51 and RPA nuclear staining. More pronounced effects were seen with a naturally occurring BRCT mutant (M1775R) that also produced elevated levels of ssDNA, in part co-localizing with RPA, in line with excessive DNA resection. M1775R induced unusual, thread-like promyelocytic leukemia (PML) nuclear bodies and clustered RPA foci rather than the typical juxtaposed RPA-PML foci seen with wild-type BRCA1. Interestingly, K1702M hyper-recombination diminished with a second mutation in the BRCA1 RING domain (I26A) known to reduce BRCA1 ubiquitin-ligase activity. Thesein vitro findings correlated with elevated nuclear RAD51 and RPA staining of breast cancer tissue from a patient with the M1775R mutation. Altogether, the disruption of BRCA1 (BRCT)-pS-X-X-F protein binding results in ubiquitination-dependent hyper-recombination via excessive DNA resection and the appearance of atypical PML-NBs. Thus, certain BRCA1 mutations that cause hyper-recombination instead of reduced DSB repair might lead to breast cancer.