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.
Bibliographical noteFunding Information:
We thank the Optical Imaging Centre for use and technical assistance with the optical microscopes; Ihor Smal (Erasmus MC) for assistance in single-molecule tracking analysis; Luke Lavis (HHMI Janelia) for providing HaloTag ligands; Niklas Bachmann for assistance in making the BRCA2-Halo DCTD cell line. We thank Joyce Lebbink (Erasmus MC) and Nick van der Zon (Erasmus MC) for critically reading the manuscript. We acknowledge the Josephine Nefkens Cancer Program for infrastructural support.
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