Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells

Cells of vertebrates remove DNA double-strand breaks (DSBs) from their geno me predominantly utilizing a fast, DNA-PKcs-dependent form of nonhomologous end joining (D-NHEJ). Mutants with inactive DNA-PKcs remove the majority o f DNA DSBs utilizing a slow, DNA-PKcs-independent pathway that does not uti lize genes of the RAD52 epistasis group, is error-prone and can therefore b e classified as a form of NHEJ (termed basic or B-NHEJ). We studied the rol e of DNA ligase IV in these pathways of NHEJ. Although biochemical studies show physical and functional interactions between the DNA-PKcs/Ku and the D NA ligase IV/Xrcc4 complexes suggesting operation within the same pathway, genetic evidence to support this notion is lacking in mammalian cells. Prim ary human fibroblasts (180BR) with an inactivating mutation in DMA ligase I V, rejoined DNA DSBs predominantly with slow kinetics similar to those obse rved in cells deficient in DNA-PKcs, or in wild-type cells treated with wor tmannin to inactivate DNA-PK. Treatment of 180BR cells with wortmannin had only a small effect on DNA DSB rejoining and no effect on cell radiosensiti vity to killing although it sensitized control cells to 180BR levels. This is consistent with DNA ligase IV functioning as a component of the D-NHEJ, and demonstrates the unperturbed operation of the DNA-PKcs-independent path way (B-NHEJ) at significantly reduced levels of DNA ligase IV. in vitro, ex tracts of 180BR cells supported end joining of restriction endonuclease-dig ested plasmid to the same degree as extracts of control cells when tested a t 10 mM Mg2+. At 0.5 mM Mg2+, where only DMA ligase IV is expected to retai n activity, low levels of end joining (similar to 10% of 10 mM) were seen i n the control but there was no detectable activity in 180BR cells. Antibodi es raised against DNA ligase IV did not measurably inhibit end joining at I O mM Mg2+ in either cell line. Thus, in contrast to the situation in vivo, end joining in vitro is dominated by pathways with properties similar to B- NHEJ that do not display a strong dependence on DNA ligase IV, with D-NHEJ retaining only a limited contribution. The implications of these observatio ns to studies of NHEJ in vivo and in vitro are discussed.