Ku70/Ku80 protein complex inhibits the binding of nucleotide excision repair proteins on linear DNA in vitro

We have previously reported that the incision efficiency of the nucleotide excision repair (NER) reaction measured in vitro with cell-free human prote in extracts was reduced by up to 80% on a Linearized damaged plasmid DNA su bstrate when compared to supercoiled damaged DNA. The inhibition stemed fro m the presence of the DNA-end binding Ku70/Ku80 heterodimer which is the re gulatory subunit of the DNA-dependent protein kinase (DNA-PK). Here, the or igin of the repair inhibition was assessed by a new in vitro assay in which circular or linear plasmid DNA, damaged or undamaged, was quantitatively a dsorbed on sensitized microplate wells. The binding of two NER proteins, XP A and p62-TFIIH, indispensable for the incision step of the reaction, was q uantified either directly in an ELISA-like reaction in the wells with speci fic antibodies or in Western blotting experiments on the DNA-bound fraction . We report a dramatic inhibition of XPA and p62-TFIIH association with WC photoproducts on linear DNA. XPA and p62-TFIIH binding to DNA damage was re gained when the reaction was performed with extracts lacking Ku activity (e xtracts from xrs6 rodent cells) whereas addition of purified human Ku compl ex to these extracts restored the inhibition. Despite the fact that DNA-PK was active during the NER reaction, the mechanism of inhibition relied on t he sole Ku complex, since mutant protein extracts lacking the catalytic DNA -PK subunit (extracts from the human M059J glioma cells) exhibited a strong binding inhibition of XPA and p62-TFIIH proteins on linear damaged DNA, id entical to the inhibition observed with the DNA-PK+ control extracts (from M059K cells). (C) 1998 Academic Press.