Interplay between human DNA repair proteins at a unique double-strand break in vivo.
DNA repair by homologous recombination is essential for preserving genomic integrity. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) play important roles in this process. In this study, we show that human RAD51 interacts with RAD51C-XRCC3 or RAD51B-C-D-XRCC2. In addition to being critical for RAD51 focus formation, RAD51C localizes to DNA damage sites. Inhibition of RAD51C results in a decrease in cellular proliferation consistent with a role in repairing double-strand breaks (DSBs) that occur naturally. To monitor a single DNA repair event, we developed immunofluorescence and chromatin immunoprecipitation (ChIP) methods on human cells where a unique DSB can be created in vivo. Using this system, we observed a single focus of RAD51C, RAD51 and 53BP1, which colocalized with gamma-H2AX. ChIPs revealed that endogenous human RAD51, RAD51C, RAD51D, XRCC2, XRCC3 and MRE11 proteins are recruited in the S-G2 phase of the cell cycle, while Ku80 is recruited during G1. We propose that RAD51C ensures a tight regulation of RAD51 assembly during DSB repair and plays a direct role in repairing DSBs in vivo.
Rodrigue
Amélie
A
Genome Stability Laboratory, Laval University Cancer Research Center, Québec City, Québec, Canada.
Lafrance
Matthieu
M
Gauthier
Marie-Christine
MC
McDonald
Darin
D
Hendzel
Michael
M
West
Stephen C
SC
Jasin
Maria
M
Masson
Jean-Yves
JY
D002453:Cell Cycle; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000032:analysis / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D051135:Rad51 Recombinase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D000071857:Tumor Suppressor p53-Binding Protein 1
D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; C448305:RAD51C protein, human; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; C110901:X-ray repair cross complementing protein 3; C113187:XRCC2 protein, human; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase