DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining

Non-homologous DNA end joining (NHEJ) is considered the major pathway of do uble-strand break (DSB) repair in mammalian cells and depends, among other things, on the DNA end-binding Ku70/80 heterodimer, To investigate the func tion of Ku in NHEJ we have compared the ability of cell-free extracts from wild-type CHO-K1 cells, Ku80-deficient xrs6 cells and Ku80-cDNA-complemente d xrs6 cells (xrs6-Ku80) to rejoin different types of DSB in vitro, While t he two Ku80-proficient extracts were highly efficient and accurate in rejoi ning all types of DNA ends, the xrs6 extract displayed strongly decreased N HEJ efficiency and accuracy, The lack of accuracy is most evident in non-ho mologous terminus configurations containing 3'-overhangs that abut a 5'-ove rhang or blunt end, While the sequences of the 3'-overhangs are mostly pres erved by fill-in DNA synthesis in the Ku80-proficient extracts, they are al ways completely lost in the xrs6 extract so that, instead, small deletions displaying microhomology patches at their break-points arise. In summary, o ur results are consistent with previous results from Ku-deficient yeast str ains and indicate that Ku may serve as an alignment factor that not only in creases NHEJ efficiency but also accuracy, furthermore, a secondary NHEJ ac tivity is present in the absence of Ku which is error-prone and possibly as sisted by base pairing interactions.