title: Effect of the BRCA2 CTRD domain on RAD51 filaments analyzed by an ensemble of single molecule techniques.
abstract: Homologous recombination is essential for the preservation of genome stability, thereby preventing cancer. The recombination protein RAD51 drives DNA strand exchange, which requires the assembly, rearrangement and disassembly of a RAD51 filament on DNA, coupled to ATP binding and hydrolysis. This process is facilitated and controlled by recombination mediators and accessory factors. Here, we have employed a range of single molecule techniques to determine the influence of the C-terminal RAD51 interaction domain (CTRD) of the breast cancer tumor suppressor BRCA2 on intrinsic aspects of RAD51-DNA interactions. We show that at high concentration the CTRD entangles RAD51 filaments and reduces RAD51 filament formation in a concentration dependent manner. It does not affect the rate of filament disassembly measured as the loss of fluorescent signal due to intrinsic RAD51 protein dissociation from double-stranded DNA (dsDNA). We conclude that, outside the context of the full-length protein, the CTRD does not reduce RAD51 dissociation kinetics, but instead hinders filament formation on dsDNA. The CTRDs mode of action is most likely sequestration of multiple RAD51 molecules thereby rendering them inactive for filament formation on dsDNA.
authors.affiliation: Department of Genetics, Cancer Genomics Center, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: J T
authors.initials: JT
authors.lastname: Holthausen
authors.forename: M T J
authors.initials: MT
authors.lastname: van Loenhout
authors.forename: H
authors.initials: H
authors.lastname: Sanchez
authors.forename: D
authors.initials: D
authors.lastname: Ristic
authors.forename: S E
authors.initials: SE
authors.lastname: van Rossum-Fikkert
authors.forename: M
authors.initials: M
authors.lastname: Modesti
authors.forename: C
authors.initials: C
authors.lastname: Dekker
authors.forename: R
authors.initials: R
authors.lastname: Kanaar
authors.forename: C
authors.initials: C
authors.lastname: Wyman
mesh_terms: D024682:BRCA2 Protein / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D054730:Protein Interaction Domains and Motifs; D051135:Rad51 Recombinase / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
chemical_list: D024682:BRCA2 Protein; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase