Human DNA ligases I and III, but not ligase IV, are required for microhomology-mediated end joining of DNA double-strand breaks.
DNA nonhomologous end-joining (NHEJ) and homologous recombination are two distinct pathways of DNA double-strand break repair in mammalian cells. Biochemical and genetic studies showed that DNA ends can also be joined via microhomology-mediated end joining (MHEJ), especially when proteins responsible for NHEJ, such as Ku, are reduced or absent. While it has been known that Ku-dependent NHEJ requires DNA ligase IV, it is unclear which DNA ligase(s) is required for Ku-independent MHEJ. In this study, we used a cell-free assay to determine the roles of DNA ligases I, III and IV in MHEJ and NHEJ. We found that siRNA mediated down-regulation of DNA ligase I or ligase III in human HTD114 cells led to impaired end joining that was mediated by 2-, 3- or 10-bp microhomology. In addition, nuclear extract from human fibroblasts harboring a mutation in DNA ligase I displayed reduced MHEJ activity. Furthermore, treatment of HTD114 nuclear extracts with an antibody against DNA ligase I or III also significantly reduced MHEJ. These data indicate that DNA ligases I and III are required in MHEJ. DNA ligase IV, on the contrary, is not required in MHEJ but facilitates Ku-dependent NHEJ. Therefore, MHEJ and NHEJ require different DNA ligases.
Liang
Li
L
Department of Genetics, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA. liang@biology.rutgers.edu
Deng
Li
L
Nguyen
Son C
SC
Zhao
Xin
X
Maulion
Christopher D
CD
Shao
Changshun
C
Tischfield
Jay A
JA
D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D004260:DNA Repair; D006801:Humans; D000072200:Ku Autoantigen; D000075223:Poly-ADP-Ribose Binding Proteins; D034622:RNA Interference; D011995:Recombination, Genetic; D029867:Xenopus Proteins
C000607892:LIG1 protein, human; C000607893:LIG4 protein, human; D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human