A possible role of Ku in mediating sequential repair of closely opposed lesions

One of the hallmarks of ionizing radiation exposure is the formation of clu stered damage that includes closely opposed lesions. We show that the Ku70/ 80 complex (Ku) has a role in the repair of closely opposed lesions in DNA. DNA containing a dihydrouracil (DHU) close to an opposing single strand br eak was used as a model substrate. It was found that gu has no effect on th e enzymatic activity of human endonuclease III when the substrate DNA conta ins only DHU, However, with DNA containing a DHU that is closely opposed to a single strand break, Ku inhibited the nicking activity of human endonucl ease III as well as the amount of free double strand breaks induced by the enzyme, The inhibition on the formation of a free double strand break by Ku was found to be much greater than the inhibition of human endonuclease III -nicking activity by Ku, Furthermore, there was a concomitant increase in t he formation of Ku-DNA complexes when endonuclease III was present. Similar results were also observed with Escherichia coli endonuclease III. These r esults suggest that Ku reduces the formation of endonuclease III-induced fr ee double strand breaks by sequestering the double strand breaks formed as a Ku-DNA complex. In doing so, Ku helps to avoid the formation of the inter mediary free double strand breaks, possibly helping to reduce the mutagenic event that might result from the misjoining of frank double strand breaks.