Abstract
Mutation of the XRCC4 gene in mammalian cells1,2 prevents the formation of the signal and coding joints in the V(D)J recombination reaction3, which is necessary for production of a functional immunoglobulin gene, and renders the cells highly sensitive to ionizing radiation4. However, XRCC4 shares no sequence homology with other proteins, nor does it have a biochemical activity to indicate what its function might be2. Here we show that DNA ligase IV (ref. 5) co-immunoprecipitates with XRCC4 and that these two proteins specifically interact with one another in a yeast two-hybrid system. Ligation of DNA double-strand breaks in a cell-free system by DNA ligase IV is increased fivefold by purified XRCC4 and seven- to eightfold when XRCC4 is co-expressed with DNA ligase IV. We conclude that the biological consequences of mutating XRCC4 are primarily due to the loss of its stimulatory effect on DNA ligase IV: the function of the XRCC4–DNA ligase IV complex may be to carry out the final steps of V(D)J recombination and joining of DNA ends.
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References
Stamato, T. D., Weinstein, R., Giaccia, A. & Mackenzie, L. Isolation of cell-cycle dependent gamma-ray sensitive Chinese hamster ovary cell. Somat. Cell Mol. Genet. 9, 165–173 (1983).
Li, Z. et al. The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell 83, 1079–1089 (1996).
Taccioli, G. E. et al. Impairment of V(D)J recombination in double-strand break repair mutants. Science 260, 207–210 (1993).
Giaccia, A. J., Richardson, E., Denko, N. & Stamato, T. D. Genetic analysis of the XR-1 mutation in hamster and human hybrids. Somatic Cell Mol. Genet. 15, 71–79 (1989).
Wei, Y. -F. et al. Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination. Mol. Cell. Biol. 15, 3206–3216 (1995).
Wilm, M. et al. Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature 379, 466–469 (1996).
Robins, P. & Lindahl, T. DNA ligase IV from HeLa cell nuclei. J. Biol. Chem. 271, 24257–24261 (1996).
Wu, X. & Lieber, M. R. Protein–protein and protein–DNA interaction regions within the DNA end binding protein Ku70–Ku86. Mol. Cell. Biol. 16, 5186–5193 (1996).
Lindahl, T. & Barnes, D. E. Mammalian DNA ligases. Annu. Rev. Biochem. 61, 251–281 (1992).
Gerstein, R. M. & Lieber, M. R. Extent to which homology can constrain coding exon junctional diversity in V(D)J recombination. Nature 363, 625–627 (1993).
Reeves, W. H. & Sthoeger, Z. M. Molecular cloning of cDNA encoding the p70(Ku) lupus autoantigen. J. Biol. Chem. 264, 5047–5052 (1989).
Mimori, T. et al. Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome. Proc. Natl Acad. Sci. USA 87, 1777–1781 (1990).
Hartley, K. O. et al. DNA-dependent protein kinase catalytic subunit: A relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell 82, 849–856 (1995).
Kirchgessner, C. U. et al. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science 267, 1178–1183 (1995).
Blunt, T. et al. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 80, 813–823 (1995).
Jackson, S. P. & Jeggo, P. A. DNA double-strand break repair and V(D)J recombination: involvement of DNA-PK. Trends Biochem. Sci. 20, 412–415 (1995).
Hsieh, C. L., Arlett, C. F. & Lieber, M. R. V(D)J recombination in ataxia telangiectasia, Bloom's syndrome, and a DNA ligase I-associated immunodeficiency disorder. J. Biol. Chem. 268, 20105–10109 (1993).
Petrini, J., Donovan, J. W., Dimare, C. & Weaver, D. T. Normal V(D)J coding junction formation in DNA ligase I deficiency syndromes. J. Immunol. 152, 176–183 (1994).
Kemp, L., Sedgwick, S. & Jeggo, P. X-ray sensitive mutants of Chinese hamster ovary cells defective in double-strand rejoining. Mut. Res. 132, 189–196 (1984).
Chen, J. et al. Mammalian DNA ligase III: molecular cloning, chromsomal localization, and expression in spermatocytes undergoing meiotic recombination. Mol. Cell. Biol. 15, 5412–5422 (1995).
Wilson, T. E., Grawunder, U. & Lieber, M. R. Yeast DNA ligase IV mediates non-homologous DNA end joining. Nature 388, 495–498 (1997).
Grawunder, U., Schatz, D. G., Leu, T. M., Rolink, A. & Melchers, F. The half-life of RAG-1 protein in precursor B cells is increased in the absence of RAG-2 expression. J. Exp. Med. 183, 1731–1737 (1996).
Lieber, M. R. et al. The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination. Cell 55, 7–16 (1988).
Gauss, G. & Lieber, M. R. DEAE–dextran enhances electroporation of mammalian cells. Nucleic Acids Res. 20, 6739–6740 (1992).
Miller, R. D. et al. Gene for the catalytic subunit of mouse DNA-PK maps to the scid locus. Proc. Natl Acad. Sci. USA 92, 10792–10795 (1995).
Mann, M. & Wilm, M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Analyt. Biochem. 66, 4390–4399 (1994).
Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. Eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl Acad. Sci. USA 83, 8122–8126 (1986).
Gyuris, J., Golemis, E., Chertkov, H. & Brent, R. Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75, 791–803 (1993).
Zervos, A., Gyuris, J. & Brent, R. Mxi1, a protein that specifically interacts with Max to bind Myc–Max recognition sites. Cell 72, 223–232 (1993).
Guarente, L. Yeast promoters and lacZ fusion designed to study expression of cloned genes in yeast. Meth. Enzymol. 101, 181–191 (1983).
Acknowledgements
U.G. is a postdoctoral fellow of the Boehringer Ingelheim Foundation. T.E.W. is a Howard Hughes Medical Institute physician postdoctoral fellow. This work was supported by grants to M.R.L. M.R.L. is a Leukemia Society of America scholar. We thank C. Hsieh, A. Kalb and M. Yaneva for comments on the manuscript.
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Grawunder, U., Wilm, M., Wu, X. et al. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 388, 492–495 (1997). https://doi.org/10.1038/41358
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DOI: https://doi.org/10.1038/41358
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