DNA ligase III as a candidate component of backup pathways of nonhomologous end joining.
Biochemical and genetic studies support the view that the majority of DNA double-strand breaks induced in the genome of higher eukaryotes by ionizing radiation are removed by two pathways of nonhomologous end joining (NHEJ) termed D-NHEJ and B-NHEJ. Whereas D-NHEJ depends on the activities of the DNA-dependent protein kinase and DNA ligase IV/XRCC4, components of B-NHEJ have not been identified. Using extract fractionation, we show that the majority of DNA end joining activity in extracts of HeLa cells derives from DNA ligase III. DNA ligase III fractionates through two columns with the maximum in DNA end joining activity and its depletion from the extract causes loss of activity that can be recovered by the addition of purified enzyme. The same fractionation protocols provide evidence for an additional factor strongly enhancing DNA end joining and shifting the product spectrum from circles to multimers. An in vivo plasmid assay shows that DNA ligase IV-deficient mouse embryo fibroblasts retain significant DNA end joining activity that can be reduced by up to 80% by knocking down DNA ligase III using RNA interference. These in vivo and in vitro observations identify DNA ligase III as a candidate component for B-NHEJ and point to additional factors contributing to NHEJ efficiency.
Wang
Huichen
H
Department of Radiation Oncology, Division of Experimental Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19122, USA.
Rosidi
Bustanur
B
Perrault
Ronel
R
Wang
Minli
M
Zhang
Lihua
L
Windhofer
Frank
F
Iliakis
George
G
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics; D004249:DNA Damage; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D000075223:Poly-ADP-Ribose Binding Proteins; D034622:RNA Interference; D029867:Xenopus Proteins
D004273:DNA, Neoplasm; C000607893:LIG4 protein, human; 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