Nuclear extracts lacking DNA-dependent protein kinase are deficient in multiple round transcription

We have compared levels of in vitro transcription in nuclear extracts from DNA-dependent protein kinase (DNA-PK)-deficient and DNA-PK-containing Chine se hamster ovary cell lines. DNA-PK-deficient cell lines are radiosensitive mutants lacking either the catalytic subunit or the 80-kDa subunit of the Ku protein regulatory component. Extracts from DNA-PK-deficient cell lines had a 2-7-fold decrease in the level of in vitro transcription when compare d with matched controls. This decrease was observed with several promoters. Transcription could be restored to either of the deficient extracts by add ition of small amounts of extract from the DNA-PK-containing cell lines. Tr anscription was not restored by addition of purified DNA-PK catalytic subun it, Ru protein, or individually purified general transcription factors. We conclude that extracts from DNA-PK-deficient cells lack a positively acting regulatory factor or a complex of factors not readily reconstituted with i ndividual proteins. We have also investigated the mechanistic defect in the deficient extracts and have found that the observed differences in transcr iption levels between Ku-positive and Ru-negative cell lines can be attribu ted solely to a greater ability of the Ru-positive nuclear extracts to carr y out secondary initiation events subsequent to the first round of transcri ption.