Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction.
The non-homologous end-joining pathway promotes direct enzymatic rejoining of DNA double-strand breaks (DSBs) and is an important determinant of genome stability in eukaryotic cells. Although previous work has shown that this pathway requires Ku, DNA-PKcs and the DNA ligase IV/XRCC4 complex, we found that these proteins alone did not promote efficient joining of cohesive-ended DNA fragments in a cell-free assay. To identify factors that were missing from the reaction, we screened fractions from HeLa cell extracts for the ability to stimulate the joining of cohesive DNA ends in a complementation assay containing other known proteins required for DNA DSB repair. We identified a factor that restored end-joining activity to the level observed in crude nuclear extracts. Factor activity copurified with Rad50, Mre11 and NBS1, three proteins that have previously been implicated in DSB repair by genetic and cytologic evidence. Factor activity was inhibited by anti-Mre11 antibody. The reconstituted system remained fully dependent on DNL IV/XRCC4 and at least partially dependent on Ku, but the requirement for DNA-PKcs was progressively lost as other components were purified. Results support a model where DNA-PKcs acts early in the DSB repair pathway to regulate progression of the reaction, and where Mre11, Rad50 and NBS1 play a key role in aligning DNA ends in a synaptic complex immediately prior to ligation.
Huang
Juren
J
Institute of Molecular Medicine and Genetics, Program in Gene Regulation, CB-2803, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA.
Dynan
William S
WS
D017766:Acid Anhydride Hydrolases; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D034961:Antigens, Nuclear; D015153:Blotting, Western; D002457:Cell Extracts; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D045643:DNA Repair Enzymes; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D019281:Dimerization; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D000076228:MRE11 Homologue Protein; D008954:Models, Biological; D009687:Nuclear Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism
D000906:Antibodies; D034961:Antigens, Nuclear; D002457:Cell Extracts; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D045643:DNA Repair Enzymes