Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre.
DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms. To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize in vivo DSBR in single cells. Using this system, we demonstrate for the first time that Rad52 DNA repair foci and DSBs colocalize. Time-lapse microscopy reveals that the relocalization of Rad52 protein into a focal assembly is a rapid and reversible process. In addition, analysis of DNA damage checkpoint-deficient cells provides direct evidence for coordination between DNA repair and subsequent release from checkpoint arrest. Finally, analyses of cells experiencing multiple DSBs demonstrate that Rad52 foci are centres of DNA repair capable of simultaneously recruiting more than one DSB.
Lisby
Michael
M
Department of Genetics & Development, Columbia University, College of Physicians & Surgeons, 701 West 168th Street, New York, NY 10032-2704, USA.
Mortensen
Uffe H
UH
Rothstein
Rodney
R
D001426:Bacterial Proteins; D004249:DNA Damage; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000502:physiology* / Q000528:radiation effects; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D016193:G1 Phase / Q000502:physiology / Q000528:radiation effects; D016195:G2 Phase / Q000502:physiology / Q000528:radiation effects; D005720:Gamma Rays / Q000009:adverse effects; D017344:Genes, Insect; D006238:Haploidy; D056968:Lac Repressors; D008938:Mitosis; D051721:Rad52 DNA Repair and Recombination Protein; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology* / Q000528:radiation effects; D013997:Time Factors
D001426:Bacterial Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific