Abstract
Purpose: To identify the protein components of the DNA end-binding complex in hamster cells.
Materials and methods: DNA end-binding complexes were identified as follows. Nuclear extracts from Chinese hamster ovary cells (0.5-1.0 µg protein lane) were incubated with 0.5 ng 32P-labelled probe (144 bp) for 20 min at room temperature in the presence of 1 µg closed circular pUC18 plasmid, a non-specific competitor in a final volume of 20 µl. The electrophoretic mobility of the protein-DNA complexes was analysed by electrophoresis in 5% polyacrylamide gels subjected to autoradiography. Antibodies to various DNA repair-associated proteins were added to the DNA end-binding complex reaction and a supershift identified DNA end-binding complex components. These were confirmed by Western analysis of purified DNA end-binding complex contents.
Results: Using both supershift and Western analysis, the following proteins were identified in the DNA end-binding complex: Ku70, Ku80, DNA-dependent protein kinase catalytic subunit, DNA ligase IV, X-ray cross complementing protein 4, meiotic recombination protein 11 (Mre11), Werner's syndrome protein, Bloom's syndrome protein, p53, poly(ADP-ribose) polymerase, replication protein A (RPA) 14, and RPA32, ataxia telangiectasia mutant, c-Abl, Rad50, Nijmegen breakage syndrome protein 1 (NBS1), and DNA ligase III were not detected in the binding complex by any assay. Using a combination of electro-elution and autoradiography, it was estimated that the single DNA end-binding complex contains at least 15 proteins whose molecular weights of the DNA end-binding proteins ranged from 620 to 12 kDa.
Conclusions: A combination of both a supershift assay and Western analysis of the DNA end-binding complexes has identified 12 of at least 15 proteins present in the DNA end-binding complex of Chinese hamster ovary cells. This protein complex contains Mre11, but not Rad50 or NBS1, suggesting that under some conditions, Mre11 might function independently of Rad50 and NBS1.