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.
Highlights
Breast cancer-associated protein 2 (BRCA2) is a required component in multi-step genome maintenance processes that are coordinated in time and place
We focused on the response of BRCA2 and RAD51 to ionizing radiation (IR)-induced DNA damage because timing of this response in wild type mouse embryonic stem (mES) cell lines is well defined, and in contrast to genotoxic chemicals, damage induction is instantaneous and synchronous
We considered that homologous recombination DNA damage response requires dynamic interaction between BRCA2 and RAD51 at a scale not evident in our cell imaging
Summary
Breast cancer-associated protein 2 (BRCA2) is a required component in multi-step genome maintenance processes that are coordinated in time and place. One role of BRCA2 common to DNA break repair, DNA crosslink repair and replication fork protection, is delivery of RAD51 to sites where it is needed (Holloman, 2011; Sharan et al, 1997; Yuan et al, 1999). RAD51 is an essential protein whose biochemical function is to form filaments on ssDNA capable of performing strand exchange reactions with homologous partners or otherwise protecting the bound. We consider essential BRCA2 activity to involve at least (1) spatial relocation in the nucleus resulting in accumulation to sites where RAD51 is needed and (2) molecular rearrangement to release or deposit RAD51 on DNA in an active form. Accumulation of the required proteins at the sites of DNA damage is typically defined as the appearance of foci, high local concentration of proteins, in immunofluorescence experiments. Several studies have addressed the role of different interactors and domains of BRCA2 on foci formation after
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
