Low levels of DNA ligases III and IV sufficient for effective NHEJ.
Cells of higher eukaryotes rejoin double strand breaks (DSBs) in their DNA predominantly by a non-homologous DNA end joining (NHEJ) pathway that utilizes the products of DNA-PKcs, Ku, LIG4, XRCC4, XLF/Cernunnos, Artemis as well as DNA polymerase lambda (termed D-NHEJ). Mutants with defects in these proteins remove a large proportion of DSBs from their genome utilizing an alternative pathway of NHEJ that operates as a backup (B-NHEJ). While D-NHEJ relies exclusively on DNA ligase IV, recent work points to DNA ligase III as a component of B-NHEJ. Here, we use RNA interference (RNAi) to further investigate the activity requirements for DNA ligase III and IV in the pathways of NHEJ. We report that 70-80% knock down of LIG3 expression has no detectable effect on DSB rejoining, either in D-NHEJ proficient cells, or in cells where D-NHEJ has been chemically or genetically compromised. Surprisingly, also LIG4 knock down has no effect on repair proficient cells, but inhibits DSB rejoining in a radiosensitive cell line with a hypomorphic LIG4 mutation that severely compromises its activity. The results suggest that complete coverage for D-NHEJ or B-NHEJ is afforded by very low ligase levels and demonstrate residual end joining by DNA ligase IV in cells of patients with mutations in LIG4.
Windhofer
Frank
F
Institute of Medical Radiation Biology, University Duisburg-Essen Medical School, Essen, Germany.
Wu
Wenqi
W
Iliakis
George
G
D000730:Androstadienes / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D010726:Phosphodiesterase Inhibitors / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D034622:RNA Interference; D000077191:Wortmannin; D029867:Xenopus Proteins
D000730:Androstadienes; C000607893:LIG4 protein, human; D010726:Phosphodiesterase Inhibitors; D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human; C000618818:Lig3 protein, mouse; D000077191:Wortmannin