Distinct RAD51 associations with RAD52 and BCCIP in response to DNA damage and replication stress.
RAD51 has critical roles in homologous recombination (HR) repair of DNA double-strand breaks (DSB) and restarting stalled or collapsed replication forks. In yeast, Rad51 function is facilitated by Rad52 and other "mediators." Mammalian cells express RAD52, but BRCA2 may have supplanted RAD52 in mediating RAD51 loading onto ssDNA. BCCIP interacts with BRCA2, and both proteins are important for RAD51 focus formation after ionizing radiation and HR repair of DSBs. Nonetheless, mammalian RAD52 shares biochemical activities with yeast Rad52, including RAD51 binding and single-strand annealing, suggesting a conserved role in HR. Because RAD52 and RAD51 associate, and RAD51 and BCCIP associate, we investigated the colocalization of RAD51 with BCCIP and RAD52 in human cells. We found that RAD51 colocalizes with BCCIP early after ionizing radiation, with RAD52 later, and there was little colocalization of BCCIP and RAD52. RAD52 foci are induced to a greater extent by hydroxyurea, which stalls replication forks, than by ionizing radiation. Using fluorescence recovery after photo bleaching, we show that RAD52 mobility is reduced to a greater extent by hydroxyurea than ionizing radiation. However, BCCIP showed no changes in mobility after hydroxyurea or ionizing radiation. We propose that BCCIP-dependent repair of DSBs by HR is an early RAD51 response to ionizing radiation-induced DNA damage, and that RAD52-dependent HR occurs later to restart a subset of blocked or collapsed replication forks. RAD52 and BRCA2 seem to act in parallel pathways, suggesting that targeting RAD52 in BRCA2-deficient tumors may be effective in treating these tumors.
Wray
Justin
J
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Cancer Research and Treatment Center, Albuquerque, New Mexico, USA.
Liu
Jingmei
J
Nickoloff
Jac A
JA
Shen
Zhiyuan
Z
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins / Q000235:genetics*; D018797:Cell Cycle Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication / Q000235:genetics*; D005354:Fibrosarcoma / Q000235:genetics; D016650:Fluorescein-5-isothiocyanate; D017930:Genes, Reporter; D006801:Humans; D007150:Immunohistochemistry; D008156:Luciferases / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics*; D010957:Plasmids; D051135:Rad51 Recombinase / Q000235:genetics*; D011839:Radiation, Ionizing; D011995:Recombination, Genetic
D000911:Antibodies, Monoclonal; C415366:BCCIP protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D008156:Luciferases; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D016650:Fluorescein-5-isothiocyanate