Role of BRCA2 DNA-binding and C-terminal domain in its mobility and conformation in DNA repair

Abstract

BRCA2 is an essential protein in genome maintenance, homologous recombination and replication fork protection. Its function includes multiple interaction partners and requires timely localization to relevant sites in the nucleus. We investigated the importance of the highly conserved DNA binding domain (DBD) and C-terminal domain (CTD) of BRCA2. We generated BRCA2 variants missing one or both domains in mouse ES cells and defined their contribution in HR function and dynamic localization in the nucleus, by single particle tracking of BRCA2 mobility. Changes in molecular architecture of BRCA2 induced by binding partners of purified BRCA2 was determined by scanning force microscopy. BRCA2 mobility and DNA damage-induced increase in the immobile fraction was largely unaffected by C-terminal deletions. The purified proteins missing CTD and/or DBD were defective in architectural changes correlating with reduced homologous recombination function in cells. These results emphasize BRCA2 activity at sites of damage beyond promoting RAD51 delivery.

DNA binding and RAD51 engagement by the BRCA2 C-terminus orchestrate DNA repair and replication fork preservation

“…As expected, cells that harbored the empty vector displayed hypersensitivity to MMC and Rucaparib (Fig. 5a), whereas expression of WT BRCA2 led to a marked enhancement of resistance to both DNA damaging agents 10,33,50,51 . However, cells expressing BRCA2 variants that harbor either the CTRB-F/A or 4 A mutation were clearly sensitized to MMC and Rucaparib, and, importantly, cells that expressed the double BRCA2 mutant exhibited higher sensitivity to these agents than either of the single mutants (Fig.…”

Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA damage repair

“…How DNA damage signals for increased interaction between DPP9 and BRCA2 remains to be shown. However, we note that the N‐terminus of BRCA2 is intrinsically disordered (Le et al , 2020 ; Sidhu et al , 2020 ; Paul et al , 2021 ) and is involved in the formation of BRCA2 dimers and multimers through self‐interactions (Shahid et al , 2014 ; Reuter et al , 2015 ; Sánchez et al , 2017 ; Le et al , 2020 ; Sidhu et al , 2020 ). Thus, it is tempting to speculate that BRCA2 is shielded from DPP9 in the multimeric form, and becomes available as a DPP9 substrate by a transition of BRCA2 multimers to monomers, a process that is favored by ssDNA, RAD51, increased temperature, and the BRCA2 chaperone DSS1 (Le et al , 2020 ; Sidhu et al , 2020 ).…”

Association of location of BRCA1 and BRCA2 mutations with benefit from olaparib and bevacizumab maintenance in high-grade ovarian cancer: phase III PAOLA-1/ENGOT-ov25 trial subgroup exploratory analysis

“…The DBD is the most conserved part of the BRCA2 protein, from fungi to humans, 41 but its function is less clear than other more 'recent' BRCA2 regions. 24 Biochemistry and microscopic studies highlighted the importance of this domain for conformation rearrangements of BRCA2 and its interaction with DNA repair partners. 24,42,43 In vitro, deletion of DBD but not the Cterminal domain of BRCA2 similarly leads to increased sensitization to platinum salts and PARPi.…”

“…24 Biochemistry and microscopic studies highlighted the importance of this domain for conformation rearrangements of BRCA2 and its interaction with DNA repair partners. 24,42,43 In vitro, deletion of DBD but not the Cterminal domain of BRCA2 similarly leads to increased sensitization to platinum salts and PARPi. 24 Studies investigating the distribution of secondary mutations of BRCA2 that restore the open reading frame of the protein, a common mechanism of secondary resistance to platinum and PARPi in patients, [44][45][46][47][48] showed that reversion mutations in BRCA2 exhibit a position dependence.…”

“…19,20 Although developed for all BRCA-inactivated tumors, 21 preclinical studies showed in genetically engineered mouse models that BRCA1 mutations located in the RING domain led to mammary tumor development characterized by reduced sensitivity to PARPi and platinum. 22,23 In vitro inactivation of the DBD of BRCA2 is associated with increased sensitivity to PARPi and platinum, 24 while large intragenic deletions in BRCA2 that completely remove the DBD but encode the TR2 domain cause PARPi resistance. 25 Anecdotal observations in clinical trials of PARPi in relapsing ovarian cancer patients selected according to their platinum sensitivity suggested that long-term responders were enriched in BRCA2 carriers 26 or BRCA structural variants.…”