Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes.
The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way.
Du
Li-Lin
LL
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Nakamura
Toru M
TM
Moser
Bettina A
BA
Russell
Paul
P
D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology; D004271:DNA, Fungal / Q000378:metabolism* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005720:Gamma Rays; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; C109867:Crb2 protein, S pombe; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C085412:rad52 protein, S pombe; D049452:Green Fluorescent Proteins; D004247:DNA; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C456840:rad3 protein, S pombe; D004720:Endonucleases; C499354:rad1 protein, S pombe; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific