Cellular redistribution of Rad51 in response to DNA damage: novel role for Rad51C.
Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Otto S
OS
Gildemeister
Jay M
JM
Sage
Kendall L
KL
Knight
D021581:Active Transport, Cell Nucleus; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D024522:Genes, BRCA2; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007259:Infrared Rays; D019913:Nuclear Localization Signals; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D019913:Nuclear Localization Signals; C448305:RAD51C protein, human; D012333:RNA, Messenger; C110901:X-ray repair cross complementing protein 3; D051135:Rad51 Recombinase