Identification of proteins in the hamster DNA end‐binding complex

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.