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A novel DNA binding domain in BRCA2 with possible implications in breast cancer risk

BRCA2 tumour suppressor protein has a recognized role in the repair of DNA double strand breaks by homologous recombination. The work of the Homologous Recombination and Cancer team describes a novel DNA binding domain in BRCA2 that can promote its recombination function and that is found mutated in breast cancer. These findings may have implications for the assessment of breast cancer risk.

Germline mutations in the breast cancer susceptibility gene BRCA2 predispose to breast, ovarian, and other cancers. BRCA2 protein has a recognized role in homologous recombination (HR), a major pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells, the defining step of which is homologous strand exchange directed by the recombinase protein RAD51.

BRCA2 promotes RAD51 recombination function by two well defined domains, the BRC repeats, that allow binding to RAD51, and the DNA binding domain located at the C-terminus (CTD). Paradoxically, BRCA2 peptides lacking the entire CTD or analysis of BRCA2 deficient cell clones that become resistant to DNA damaging agents, missing this region, result in an unexpected mild phenotype and/or partial restoration of the HR proficiency suggesting another domain may take over CTD’s function. So far the existence of this domain has remained elusive.

Schematic representation of DNA damage repair mediated by the N-terminal DNA binding domain of BRCA2. Upon DNA damage, the N-terminus of BRCA2 (NTD) is positioned at the dsDNA/ssDNA junction of a resected DNA break. The recombinase protein RAD51 forms a filament on ssDNA and catalyzes the strand exchange reaction with the intact copy of the chromosome to repair the break. A BRCA2 mutation found in breast cancer reduces the DNA binding activity of the NTD precluding the repair of the break.

In this work we reveal a novel DNA binding domain in BRCA2 located at its N-terminal region (NTD). Unexpectedly, the NTD binds with stronger affinity to DNA than the CTD, and in contrast to it, it binds readily to dsDNA. This dsDNA binding activity is required to stimulate RAD51-strand exchange activity in vitro when using dsDNA/ssDNA containing substrates, the type of DNA substrate generated upon DSBs. To assess the potential clinical significance of these data, we examined the Breast Cancer Information Core (BIC) and BRCAshare databases to identify BRCA2 sequence polymorphisms that mapped to the NTD region. Importantly, a mutation of BRCA2 identified several times in breast cancer but of unknown clinical significance located in the NTD region reduces both the dsDNA binding and the recombination activity of RAD51 at dsDNA/ssDNA containing substrates.

This work provides a molecular explanation for the mild DNA damage phenotype observed in cells devoid of CTD of BRCA2, sheds light on the functional relevance of the highly unstructured N-terminal region of BRCA2 and have implications for the evaluation of BRCA2 variants identified in breast cancer patients.

Original Article:
A second DNA binding site in human BRCA2 promotes homologous recombination

Special thank to Ahmed El Marjou and Patricia Duchambon of the Protein Production and Purification Facility for their help.