Tracking Ku antigen levels in cell extracts with DNA containing abasic sites.

摘要

Prominent lesions in DNA are abasic (AP) sites arising spontaneously or as intermediates during base excision repair. An AP site can form a Schiff base intermediate with primary amino groups of proteins. This intermediate can be stabilized by NaBH(4) treatment and, therefore, cross-linking of AP site-containing DNA (AP DNA) can be used as a tool in detecting proteins that interact with AP sites. Using AP DNA, we observed in the extracts derived from several human cell lines a predominant cross-linked product with an apparent molecular mass of 95kDa. The cross-linked protein was identified as the p80 subunit of Ku antigen (Ku80) (Ilina et al., Biochem. Biophys. Acta 1784 (2008) 1777-1785 [1]). Because the cross-linking of Ku80 to AP sites is efficient and selective, this approach may be useful to estimate the amount of Ku antigen in cell extracts in the presence of other cellular proteins. We compared levels of Ku80 detected by dot-ELISA with Ku80 antibodies to the levels of Ku80 cross-linked to AP DNA in extracts derived from HeLa cells and several melanoma cell lines. The level of Ku80 trapping varied considerably depending on the cell lines and correlated with the amount of Ku80 in the extracts estimated by the immunochemical approach. This approach, unlike western blot or estimation of the Ku content based on mRNA levels, is more suitable for tracking Ku forms active in DNA binding including those having aberrations in Ku80, but retaining an ability to heterodimerize with Ku70, that provides efficient loading of Ku antigen onto DNA ends. As a routine test, borohydride trapping (BHT) is also less time and reagent consuming than blotting and EMSA.