Accurate in vitro end joining of a DNA double strand break with partially cohesive 3 '-overhangs and 3 '-phosphoglycolate termini - Effect of Ku on repair fidelity

To examine determinants of fidelity in DNA end joining, a substrate contain ing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3'-overhangs and a one-base gap in eac h strand, was constructed. In extracts of Xenopus eggs, human lymphoblastoi d cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate rest oration of the original sequence. In extracts of the Ku-deficient CHO deriv ative xrs6, a shorter product, consistent with 3' --> 5' resection before l igation, was formed. Similar results were seen for a substrate with 5'-over hangs and recessed S'-phosphoglycolate ends. Supplementation of the xrs6 ex tracts with purified Ku restored accurate end joining. In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were block ed by wortmannin, consistent with a requirement for DNA-PKcs activity. The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can acc urately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maint aining the alignment of short partial complementarities during gap filling and ligation.