Mammalian cells require non-homologous end joining (NHEJ) for the efficient repair of chromosomal DNA double-strand breaks1. A key feature of biological sources of strand breaks is associated nucleotide damage, including base loss (abasic or apurinic/apyrimidinic (AP) sites)2. At single-strand breaks, 5′-terminal abasic sites are excised by the 5′-deoxyribose-5-phosphate (5′-dRP) lyase activity of DNA polymerase β (pol β)3,4,5,6: here we show, in vitro and in cells, that accurate and efficient repair by NHEJ of double-strand breaks with such damage similarly requires 5′-dRP/AP lyase activity. Classically defined NHEJ is moreover uniquely effective at coupling this end-cleaning step to joining in cells, helping to distinguish this pathway from otherwise robust alternative NHEJ pathways. The NHEJ factor Ku can be identified as an effective 5′-dRP/AP lyase. In a similar manner to other lyases7, Ku nicks DNA 3′ of an abasic site by a mechanism involving a Schiff-base covalent intermediate with the abasic site. We show by using cell extracts that Ku is essential for the efficient removal of AP sites near double-strand breaks and, consistent with this result, that joining of such breaks is specifically decreased in cells complemented with a lyase-attenuated Ku mutant. Ku had previously been presumed only to recognize ends and recruit other factors that process ends; our data support an unexpected direct role for Ku in end-processing steps as well.
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We thank M. Gellert, T. Kunkel, M. Garcia-Diaz, T. Traut, L. Harrison and K. Meek for helpful comments. This work was supported by Public Health Service (PHS) grant CA 84442 and a Leukemia and Lymphoma Society scholar award to D.A.R., and by PHS grants R01 CA76317-05A1 and P01 AG17242 to P.H.
Author Contributions Experiments were designed by S.A.R. and D.A.R. In vitro experiments were performed by S.A.R, N.S., M.D.B. and D.A.R. Mutagenesis and protein purification were performed by S.A.R. and D.A.R. S.A.R., C.S., J.M.H. and M.D.B. performed cellular experiments. P.H. provided Ku70 knockout dermal fibroblasts. S.A.R. and D.A.R. wrote the manuscript with the aid of N.S. and M.D.B.
The authors declare no competing financial interests.
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Roberts, S., Strande, N., Burkhalter, M. et al. Ku is a 5′-dRP/AP lyase that excises nucleotide damage near broken ends. Nature 464, 1214–1217 (2010). https://doi.org/10.1038/nature08926
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