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

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

eLife (2021-07-14)
Maarten W Paul, Arshdeep Sidhu, Yongxin Liang, Sarah E van Rossum-Fikkert, Hanny Odijk, Alex N Zelensky, Roland Kanaar, Claire Wyman
ABSTRACT

Breast cancer type two susceptibility protein (BRCA2) is an essential protein in genome maintenance, homologous recombination (HR), 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 embryonic stem (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 were determined by scanning force microscopy. BRCA2 mobility and DNA-damage-induced increase in the immobile fraction were largely unaffected by C-terminal deletions. The purified proteins missing CTD and/or DBD were defective in architectural changes correlating with reduced HR function in cells. These results emphasize BRCA2 activity at sites of damage beyond promoting RAD51 delivery.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-BRCA2 (Ab-1) Mouse mAb (2B), liquid, clone 2B, Calbiochem®
Sigma-Aldrich
Anti-Rabbit IgG (H+L), CF568 F(ab′)2 fragment of antibody produced in goat, ~2 mg/mL, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
cis-Diammineplatinum(II) dichloride, crystalline