Interaction between BRCA2 and replication protein A is compromised by a cancer-predisposing mutation in BRCA2

Abstract

Mutations in the BRCA1 and BRCA2 genes predispose women to familial, early-onset breast cancer. Both the BRCA1 and BRCA2 proteins appear to function in the homologous recombination pathway of DNA doublestrand break repair. Both BRCA1 and BRCA2 have also been implicated in transcription by RNA polymerase II, for both proteins have domains which, when tethered adjacent to a promoter, can activate transcription. In experiments reported here, we have used protein affinity chromatography and coimmunoprecipitation techniques to show that the putative N-terminal acidic transcriptional activation domain of BRCA2 interacts with replication protein A (RPA), a protein essential for DNA repair, replication and recombination. This interaction was not mediated by DNA and was specific for human RPA but not yeast RPA. Since the cancer-predisposing mutation Y42C in BRCA2 significantly compromised the interaction between RPA and BRCA2, this interaction may be biologically important. That BRCA2 protein in HeLa cell extract also coimmunoprecipitated with RPA suggested that this interaction occurs in vivo. Therefore, the transcriptional activation domains within BRCA2, and perhaps BRCA1, may provide links to RPA and DNA repair processes rather than transcription.

BRCA1 and BRCA2 germline mutation spectrum and frequencies in Belgian breast/ovarian cancer families

“…Only BRCA2 Y42C was thought to be a pathogenic mutation. Y42 is a highly conserved amino acid and Y to C is a radical amino-acid change, compromising in vivo the interaction between BRCA2 and replication protein A (Wong et al, 2003). All splice site mutations were studied at the RNA level Claes et al, 2003).…”

Sequential and Synergistic Modification of Human RPA Stimulates Chromosomal DNA Repair

“…Olson et al (24) have concluded that Ser 33 is modified by ATR. The remaining sites (Ser 4 , Ser 8 , Ser 11 , Ser 12 , and Ser 13 ) are phosphorylated in response to genotoxic stress, although the responsible kinase(s) in vivo has not yet been identified. However, all can be modified by DNA-PK in vitro (22).…”

“…(That is, the initial modification of Ser 33 by ATR stimulates subsequent phosphorylation of Cdk sites Ser 23 and Ser 29 ). These events then facilitate modification of Thr 21 and extreme N-terminal sites Ser 4 and Ser 8 , probably by DNA-PK. Our data also indicate that the phosphorylation of one RPA molecule can influence the phosphorylation of other RPA molecules in trans.…”

“…For DNA replication, the study of cellular and viral model systems demonstrates that RPA is needed both for origin denaturation and replication elongation, in the latter case to facilitate the switch from DNA polymerase ␣ to DNA polymerase ␦ during Okazaki fragment synthesis (3). RPA acts in homologous recombination (HR) to stimulate DNA annealing using physical interactions with Rad52 (4 -7) and in HR-mediated DNA repair, probably employing specific interactions with BRCA2 (8,9). RPA is a required factor in both the nucleotide excision (10,11) and mismatch repair pathways (12,13) and in somatic hypermutation (14).…”

A network medicine approach to human disease

“…Most of the considered mutations showed a LOF by mainly disrupting transient protein-protein interactions. For instance, a mutation in BRCA2, a breast cancer related gene, impairs the interaction with the replication protein A complex, essential for DNA repair [24], which leads to an accumulation of carcinogenic DNA damages. Another case of a LOF mutation occurs in the von Hippel-Lindau (VHL) tumor suppressor gene involved in the VHL syndrome, an inherited predisposition to a variety of cancer types [25].…”