Ku is a 5dRP/AP lyase that excises nucleotide damage near broken ends

摘要

Mammalian cells require Nonhomologous end joining (NHEJ) for efficient repair of chromosomal DNA double-strand breaks. A key feature of biological sources of strand breaks is associated nucleotide damage, including base loss (abasic or AP sites). At single strand breaks, 5' terminal abasic sites are excised by pol β's 5'dRP lyase activity^,,,: we show here 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 distinguish this pathway from otherwise robust alternate NHEJ pathways. Surprisingly, the NHEJ factor Ku can be identified as an effective 5'dRP/AP lyase. Similar to other lyases, Ku nicks DNA 3' of an abasic site by a mechanism involving a Schiff base covalent intermediate with the abasic site. We demonstrate using cell extracts that Ku is essential for efficient removal of AP sites near double-strand breaks and, consistent with this result, joining of such breaks is specifically reduced in cells complemented with a lyase-attenuated Ku mutant. Ku had previously been presumed only to recognize ends and recruit other factors that processed ends; our data supports an unexpected direct role for Ku in end processing steps as well.NHEJ of ends with abasic sites. Substrate cartoons show position of abasic site (open circle; if reduced, closed circle) relative to intact nucleotides (blocks/letters). >a, Repair of strand breaks (single strand, SSB; double-strand, DSB) with associated abasic sites. >b, Radiolabeled 250 bp substrates were incubated with purified Ku, DNA-PKcs, XRCC4-LigaseIV, and XLF at 37°C for 20 minutes (5'dRP-EJ) or 60 minutes (AP-EJ) and products detected by electrophoresis and phosphorimaging. R; abasic sites reduced. >c and >d, Substrates with end structures varied as noted in cartoons were introduced into Ku70^−/− fibroblasts complemented with Ku70 or empty vector standard. Recovered DNA was then assessed for efficiencies of substrate recovery and joining by qPCRs using substrate and junction primer pairs, respectively. Error bars reflect the standard error of the mean (s.e.m.) from 4 independent transfections.