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

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. These in 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.

BRCA1-directed, enhanced and aberrant homologous recombination

“…On the other hand, silencing BRCA1, BRIP1 or CtIP resulted in decreased HR, suggesting that HHR is dependent on the presence of BRCA1, and that the disruption of interactions within the BRCA1-A complex is the critical step resulting in these effects. 24,25 Together, these three reports concluded that disruption of BRCT interactions, whether resulting from mutations in the BRCT domain or from specifically preventing the A complex from binding to the BRCT domain, resulted in HHR [24][25][26] ( Fig. 1A and C).…”

“…Two reports examined the effects of silencing BRCT-interacting proteins, while a third examined the effect of mutating the binding pocket of the BRCT domain to prevent these interactions, with all reaching similar conclusions. [24][25][26] Silencing Abraxas, RAP80 or the BRCC36 deubiquitinase, all part of the A complex, resulted in increased or hyper-HR (HHR). Similarly, synthetic (K1702M) and naturally occurring (M1775R) mutants of BRCA1 that disrupt most, if not all, BRCT pS-X-X-F-binding interactions also resulted in aberrant HHR.…”

DNA damage and breast cancer

“…However, the process of DNA repair itself can create mutations, insertions, deletions and base replacements. For example, mutations can occur due to inappropriately high levels of a repair mechanism which normally suppresses tumorigenesis-mitotic recombination and hyper-recombinationwhich has recently been linked to mutations in BRCA1 but is also likely to occur due to other defects in DNA damage repair genes [28][29][30] . Gross DNA lesions and chromosomal abnormalities can be induced by DNA replication stress.…”

“…One important gene [59] ; and (2) an N-terminal RING domain which interacts with BARD1 protein to allow for E3 ubiquitinligase activity [60][61][62] . The BRCT domain contains repeats which bind to unique phosphor-serine motifs of other proteins and which allow participation in DNA damage checkpoint and double strand break repairs [30,42,59,63] . BRCT domains are also found in other breast-cancerrelated genes such as XRCC4 or FANCG.…”

“…It is normative for these gene products to form protein complexes that can mediate repair of DNA damage [10,64,65] . Mutations in the BRCT domain of BRCA1 which inactivate its binding capacity, can result in increased single stranded DNA and hyper-recombination rates without changes to nonhomologous end-joining DNA repair mechanisms [30] . It is currently unknown whether similar mutations in BRCT regions of other genes may also result in hyper-recombination effects.…”