Stabilization of RAD-51-DNA filaments via an interaction domain in Caenorhabditis elegans BRCA2.
Mutations in BRCA2 predispose individuals to breast cancer, a consequence of the role of BRCA2 in DNA repair. Human BRCA2 interacts with the recombinase RAD51 via eight BRC repeats. Controversy has existed, however, about whether the BRC interactions are primarily with RAD51 monomers or with the RAD51-DNA helical polymer, and whether there is a single interaction or multiple ones. We show here that the single BRC motif in the Caenorhabditis elegans BRCA2 homolog, CeBRC-2, contains two different RAD-51-binding regions. One of these regions binds only weakly to RAD-51-DNA filaments but strongly to RAD-51 alone and corresponds to the part of human BRC4 crystallized with RAD51. Injection of a peptide corresponding to this region into worms inhibits the normal formation of RAD-51 foci in response to ionizing radiation (IR). Conversely, peptides corresponding to the second region bind strongly to RAD-51-DNA filaments but do not bind to RAD-51 alone. Three-dimensional reconstructions from electron micrographs show that this peptide binds to the RAD-51 N-terminal domain, which has been shown to have a regulatory function. Injection of this peptide into worms before IR leads to a dramatic increase and persistence of IR-induced RAD-51 foci. This peptide also inhibits the RAD-51 ATPase activity, required for filament depolymerization. These results support a model where an interaction with RAD-51 alone is likely involved in filament nucleation, whereas a second independent interaction is involved in stabilization of RAD-51 filaments by BRCA2. The multiple interactions between BRCA2-like molecules and RAD51 provide insights into why mutations in BRCA2 lead to cancer.
Petalcorin
Mark I R
MI
Clare Hall Laboratories, London Research Institute, Cancer Research UK, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
Galkin
Vitold E
VE
Yu
Xiong
X
Egelman
Edward H
EH
Boulton
Simon J
SJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D024682:BRCA2 Protein / Q000737:chemistry* / Q000378:metabolism*; D017173:Caenorhabditis elegans / Q000378:metabolism*; D018509:DNA, Helminth / Q000378:metabolism* / Q000648:ultrastructure; D005799:Genes, Dominant; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051135:Rad51 Recombinase / Q000378:metabolism* / Q000648:ultrastructure; D013329:Structure-Activity Relationship; D013997:Time Factors
D024682:BRCA2 Protein; D018509:DNA, Helminth; D000255:Adenosine Triphosphate; D051135:Rad51 Recombinase