Nucleic Acids Research, 2002, Vol. 30, No. 3 667-674
© 2002 Oxford University Press
Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction
Institute of Molecular Medicine and Genetics, Program in Gene Regulation, CB-2803, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA
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.
* To whom correspondence should be addressed. Tel: +1 706 721 8756; Fax: +1 706 721 8752; Email: wdynan{at}mail.mcg.edu Present address: Juren Huang, USC Pathology Reference Laboratory, University of Southern California, 2250 Alcazar Street, Room 109, Los Angeles, CA 90033, USA
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