DNA ligase III as a candidate component of backup pathways of nonhomologous end joining

Wang H, Rosidi B, Perrault R, Wang M, Zhang L, Windhofer F and Iliakis G
Source: Cancer Res
Publication Date: (2005)
Issue: 65(10): 4020-4030
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Embryonic fibroblast, mouse (MEF) immort
Species: mouse
Tissue Origin: embryo
Nucleofector® I/II/2b
Nucleofected linearised pEGFP plasmid into LIG4-/- MEF in order to measure DNA ligase IV independent double-strand break repair. (co-transfected with pDsRed2-N1 in order to measure transfection efficiency) transfected LIG4-/- MEF with siRNA against DNA ligase III (or control siRNA) and 24 hours later transfected same cells with linearised pEGFP in order to assess contribution of DNA ligase III to DNA ligase IV independent DSB repair.
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