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. 2001 Apr 15;29(8):1653-60.
doi: 10.1093/nar/29.8.1653.

V体育平台登录 - Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells

H Wang  1 , Z C Zeng, A R Perrault, X Cheng, W Qin, G Iliakis
Affiliations

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

H Wang et al. Nucleic Acids Res. .

Abstract

Cells of vertebrates remove DNA double-strand breaks (DSBs) from their genome predominantly utilizing a fast, DNA-PKcs-dependent form of non-homologous end joining (D-NHEJ). Mutants with inactive DNA-PKcs remove the majority of DNA DSBs utilizing a slow, DNA-PKcs-independent pathway that does not utilize genes of the RAD52 epistasis group, is error-prone and can therefore be classified as a form of NHEJ (termed basic or B-NHEJ). We studied the role of DNA ligase IV in these pathways of NHEJ. Although biochemical studies show physical and functional interactions between the DNA-PKcs/Ku and the DNA ligase IV/Xrcc4 complexes suggesting operation within the same pathway, genetic evidence to support this notion is lacking in mammalian cells. Primary human fibroblasts (180BR) with an inactivating mutation in DNA ligase IV, rejoined DNA DSBs predominantly with slow kinetics similar to those observed in cells deficient in DNA-PKcs, or in wild-type cells treated with wortmannin to inactivate DNA-PK. Treatment of 180BR cells with wortmannin had only a small effect on DNA DSB rejoining and no effect on cell radiosensitivity 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 pathway (B-NHEJ) at significantly reduced levels of DNA ligase IV. In vitro, extracts of 180BR cells supported end joining of restriction endonuclease-digested plasmid to the same degree as extracts of control cells when tested at 10 mM Mg(2+). At 0 VSports手机版. 5 mM Mg(2+), where only DNA ligase IV is expected to retain activity, low levels of end joining ( approximately 10% of 10 mM) were seen in the control but there was no detectable activity in 180BR cells. Antibodies raised against DNA ligase IV did not measurably inhibit end joining at 10 mM Mg(2+) 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 observations to studies of NHEJ in vivo and in vitro are discussed. .

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Figures

Figure 1
Figure 1
Rejoining of IR-induced DNA DSBs in 180BR primary fibroblasts in the presence or absence of 20 µM wortmannin. The upper panel shows a typical gel, while the lower panel shows the quantitative analysis from three independent experiments. Plotted are the mean and the standard error. The lines through the data points represent fitting to the sum of two exponential functions as described in Materials and Methods. The value of FAR measured in non-irradiated cells has been subtracted from all data points.
Figure 2
Figure 2
Rejoining of IR-induced DNA DSBs in MRC5 primary fibroblasts in the presence or absence of 20 µM wortmannin. Other details as in Figure 1.
Figure 3
Figure 3
Cell survival in 180BRM and MRC5sv cells after exposure to various doses of X-rays. Cells were irradiated in the presence or absence of 20 µM wortmannin, added 1 h before exposure, and plated for colony formation 4 h later. Results represent the mean and standard error from three independent experiments.
Figure 4
Figure 4
End joining after incubation at 25°C for 1 h of SalI-digested plasmid DNA by 10 µg whole cell extract prepared from actively growing 180BRM or MRC5sv cells. (A) Reactions assembled at different Mg2+ concentrations as indicated. (B) Reactions assembled either at 10 or 0.5 mM Mg2+ with extracts pre-treated for 1 h at 4°C with an anti-ligase IV monoclonal antibody as indicated. Reactions were stopped by adding 2 µl 5% SDS, 2 µl 0.5 M EDTA and 1 µl 1 mg/ml proteinase K, and incubated at 37°C for 1 h before assaying by gel electrophoresis. Gels were stained with SYBR Gold and scanned in a FluorImager. Control reactions were assembled in the absence of cell extract.
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"VSports" References

    1. DiBiase S.J., Zeng,Z.-C., Chen,R., Hyslop,T., Curran,W.J.,Jr and Iliakis,G. (2000) DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus. Cancer Res., 60, 1245–1253. - PubMed
    1. Thacker J. (1999) A surfeit of RAD51-like genes? Trends Genet., 15, 166–168. - PubMed
    1. Thompson, L.H. and Schild,D. (1999) The contribution of homologous recombination in preserving genome integrity in mammalian cells. Biochimie, 81, 87–105. - PubMed (V体育ios版)
    1. Jeggo P.A. (1998) DNA breakage and repair. Adv. Genet., 38, 186–218. - PubMed
    1. Wang H., Zeng,Z.-C., Bui,T.-A., Sonoda,E., Takata,M., Takeda,S. and Iliakis,G. (2001) Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group. Oncogene, in press. - PubMed

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