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PMID: 11809878 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction.

Nucleic acids research ·Vol. 30 ·No. 3 ·2002-02-01 ·Pages 667-74

Huang J, Dynan WS

Abstract

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.

MeSH Terms
Acid Anhydride Hydrolases Antibodies/immunology,isolation & purification,pharmacology Antigens, Nuclear Blotting, Western Cell Extracts DNA/chemistry,genetics,metabolism DNA Damage DNA Helicases DNA Repair DNA Repair Enzymes DNA-Activated Protein Kinase DNA-Binding Proteins/antagonists & inhibitors,chemistry,immunology,isolation & purification,metabolism Dimerization Genetic Complementation Test HeLa Cells Humans Ku Autoantigen MRE11 Homologue Protein Models, Biological Nuclear Proteins/chemistry,isolation & purification,metabolism Nucleic Acid Conformation Protein Serine-Threonine Kinases/isolation & purification,metabolism
Chemicals
Antibodies Antigens, Nuclear Cell Extracts DNA-Binding Proteins MRE11 protein, human Nuclear Proteins DNA DNA-Activated Protein Kinase PRKDC protein, human Protein Serine-Threonine Kinases MRE11 Homologue Protein Acid Anhydride Hydrolases Rad50 protein, human DNA Helicases XRCC5 protein, human Xrcc6 protein, human Ku Autoantigen DNA Repair Enzymes
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Huang Juren
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
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
1362-4962
DOI
10.1093/nar/30.3.667
Published
2002-02-01
Pages
667-74
Language
English
Region
England
NLM ID
0411011
PMCID
PMC100305
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/30.3.667 PMC Full Text View journal details
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