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. 2003 Dec;165(4):1929–1941. doi: 10.1093/genetics/165.4.1929

The Drosophila melanogaster DNA Ligase IV gene plays a crucial role in the repair of radiation-induced DNA double-strand breaks and acts synergistically with Rad54.

Marcin M Gorski 1, Jan C J Eeken 1, Anja W M de Jong 1, Ilse Klink 1, Marjan Loos 1, Ron J Romeijn 1, Bert L van Veen 1, Leon H Mullenders 1, Wouter Ferro 1, Albert Pastink 1
PMCID: PMC1462910  PMID: 14704177

Abstract

DNA Ligase IV has a crucial role in double-strand break (DSB) repair through nonhomologous end joining (NHEJ). Most notably, its inactivation leads to embryonic lethality in mammals. To elucidate the role of DNA Ligase IV (Lig4) in DSB repair in a multicellular lower eukaryote, we generated viable Lig4-deficient Drosophila strains by P-element-mediated mutagenesis. Embryos and larvae of mutant lines are hypersensitive to ionizing radiation but hardly so to methyl methanesulfonate (MMS) or the crosslinking agent cis-diamminedichloroplatinum (cisDDP). To determine the relative contribution of NHEJ and homologous recombination (HR) in Drosophila, Lig4; Rad54 double-mutant flies were generated. Survival studies demonstrated that both HR and NHEJ have a major role in DSB repair. The synergistic increase in sensitivity seen in the double mutant, in comparison with both single mutants, indicates that both pathways partially overlap. However, during the very first hours after fertilization NHEJ has a minor role in DSB repair after exposure to ionizing radiation. Throughout the first stages of embryogenesis of the fly, HR is the predominant pathway in DSB repair. At late stages of development NHEJ also becomes less important. The residual survival of double mutants after irradiation strongly suggests the existence of a third pathway for the repair of DSBs in Drosophila.

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Selected References

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  1. Bailey S. M., Meyne J., Chen D. J., Kurimasa A., Li G. C., Lehnert B. E., Goodwin E. H. DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. Proc Natl Acad Sci U S A. 1999 Dec 21;96(26):14899–14904. doi: 10.1073/pnas.96.26.14899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes D. E., Stamp G., Rosewell I., Denzel A., Lindahl T. Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr Biol. 1998 Dec 17;8(25):1395–1398. doi: 10.1016/s0960-9822(98)00021-9. [DOI] [PubMed] [Google Scholar]
  3. Boulton S. J., Jackson S. P. Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing. EMBO J. 1998 Mar 16;17(6):1819–1828. doi: 10.1093/emboj/17.6.1819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boulton S. J., Jackson S. P. Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. Nucleic Acids Res. 1996 Dec 1;24(23):4639–4648. doi: 10.1093/nar/24.23.4639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boulton S. J., Jackson S. P. Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways. EMBO J. 1996 Sep 16;15(18):5093–5103. [PMC free article] [PubMed] [Google Scholar]
  6. Chai Weihang, Ford Lance P., Lenertz Lisa, Wright Woodring E., Shay Jerry W. Human Ku70/80 associates physically with telomerase through interaction with hTERT. J Biol Chem. 2002 Oct 10;277(49):47242–47247. doi: 10.1074/jbc.M208542200. [DOI] [PubMed] [Google Scholar]
  7. Chevray P. M., Nathans D. Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5789–5793. doi: 10.1073/pnas.89.13.5789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clever B., Interthal H., Schmuckli-Maurer J., King J., Sigrist M., Heyer W. D. Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins. EMBO J. 1997 May 1;16(9):2535–2544. doi: 10.1093/emboj/16.9.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988 Nov 25;16(22):10881–10890. doi: 10.1093/nar/16.22.10881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Essers J., Hendriks R. W., Swagemakers S. M., Troelstra C., de Wit J., Bootsma D., Hoeijmakers J. H., Kanaar R. Disruption of mouse RAD54 reduces ionizing radiation resistance and homologous recombination. Cell. 1997 Apr 18;89(2):195–204. doi: 10.1016/s0092-8674(00)80199-3. [DOI] [PubMed] [Google Scholar]
  11. Essers J., van Steeg H., de Wit J., Swagemakers S. M., Vermeij M., Hoeijmakers J. H., Kanaar R. Homologous and non-homologous recombination differentially affect DNA damage repair in mice. EMBO J. 2000 Apr 3;19(7):1703–1710. doi: 10.1093/emboj/19.7.1703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Featherstone C., Jackson S. P. Ku, a DNA repair protein with multiple cellular functions? Mutat Res. 1999 May 14;434(1):3–15. doi: 10.1016/s0921-8777(99)00006-3. [DOI] [PubMed] [Google Scholar]
  13. Feeney A. J. Predominance of VH-D-JH junctions occurring at sites of short sequence homology results in limited junctional diversity in neonatal antibodies. J Immunol. 1992 Jul 1;149(1):222–229. [PubMed] [Google Scholar]
  14. Frank K. M., Sekiguchi J. M., Seidl K. J., Swat W., Rathbun G. A., Cheng H. L., Davidson L., Kangaloo L., Alt F. W. Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature. 1998 Nov 12;396(6707):173–177. doi: 10.1038/24172. [DOI] [PubMed] [Google Scholar]
  15. Frank K. M., Sharpless N. E., Gao Y., Sekiguchi J. M., Ferguson D. O., Zhu C., Manis J. P., Horner J., DePinho R. A., Alt F. W. DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway. Mol Cell. 2000 Jun;5(6):993–1002. doi: 10.1016/s1097-2765(00)80264-6. [DOI] [PubMed] [Google Scholar]
  16. Gao Y., Chaudhuri J., Zhu C., Davidson L., Weaver D. T., Alt F. W. A targeted DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination. Immunity. 1998 Sep;9(3):367–376. doi: 10.1016/s1074-7613(00)80619-6. [DOI] [PubMed] [Google Scholar]
  17. Gao Y., Sun Y., Frank K. M., Dikkes P., Fujiwara Y., Seidl K. J., Sekiguchi J. M., Rathbun G. A., Swat W., Wang J. A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis. Cell. 1998 Dec 23;95(7):891–902. doi: 10.1016/s0092-8674(00)81714-6. [DOI] [PubMed] [Google Scholar]
  18. Ghabrial A., Ray R. P., Schüpbach T. okra and spindle-B encode components of the RAD52 DNA repair pathway and affect meiosis and patterning in Drosophila oogenesis. Genes Dev. 1998 Sep 1;12(17):2711–2723. doi: 10.1101/gad.12.17.2711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Golub E. I., Kovalenko O. V., Gupta R. C., Ward D. C., Radding C. M. Interaction of human recombination proteins Rad51 and Rad54. Nucleic Acids Res. 1997 Oct 15;25(20):4106–4110. doi: 10.1093/nar/25.20.4106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gravel S., Larrivée M., Labrecque P., Wellinger R. J. Yeast Ku as a regulator of chromosomal DNA end structure. Science. 1998 May 1;280(5364):741–744. doi: 10.1126/science.280.5364.741. [DOI] [PubMed] [Google Scholar]
  21. Grawunder U., Wilm M., Wu X., Kulesza P., Wilson T. E., Mann M., Lieber M. R. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature. 1997 Jul 31;388(6641):492–495. doi: 10.1038/41358. [DOI] [PubMed] [Google Scholar]
  22. Grawunder U., Zimmer D., Fugmann S., Schwarz K., Lieber M. R. DNA ligase IV is essential for V(D)J recombination and DNA double-strand break repair in human precursor lymphocytes. Mol Cell. 1998 Oct;2(4):477–484. doi: 10.1016/s1097-2765(00)80147-1. [DOI] [PubMed] [Google Scholar]
  23. Grawunder U., Zimmer D., Lieber M. R. DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains. Curr Biol. 1998 Jul 16;8(15):873–876. doi: 10.1016/s0960-9822(07)00349-1. [DOI] [PubMed] [Google Scholar]
  24. Gu Y., Seidl K. J., Rathbun G. A., Zhu C., Manis J. P., van der Stoep N., Davidson L., Cheng H. L., Sekiguchi J. M., Frank K. Growth retardation and leaky SCID phenotype of Ku70-deficient mice. Immunity. 1997 Nov;7(5):653–665. doi: 10.1016/s1074-7613(00)80386-6. [DOI] [PubMed] [Google Scholar]
  25. Göttlich B., Reichenberger S., Feldmann E., Pfeiffer P. Rejoining of DNA double-strand breaks in vitro by single-strand annealing. Eur J Biochem. 1998 Dec 1;258(2):387–395. doi: 10.1046/j.1432-1327.1998.2580387.x. [DOI] [PubMed] [Google Scholar]
  26. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  27. Hsu H. L., Gilley D., Blackburn E. H., Chen D. J. Ku is associated with the telomere in mammals. Proc Natl Acad Sci U S A. 1999 Oct 26;96(22):12454–12458. doi: 10.1073/pnas.96.22.12454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jeggo P. A., Concannon P. Immune diversity and genomic stability: opposite goals but similar paths. J Photochem Photobiol B. 2001 Dec 31;65(2-3):88–96. doi: 10.1016/s1011-1344(01)00243-3. [DOI] [PubMed] [Google Scholar]
  29. Kabotyanski E. B., Gomelsky L., Han J. O., Stamato T. D., Roth D. B. Double-strand break repair in Ku86- and XRCC4-deficient cells. Nucleic Acids Res. 1998 Dec 1;26(23):5333–5342. doi: 10.1093/nar/26.23.5333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Karanjawala Zarir E., Adachi Noritaka, Irvine Ryan A., Oh Eui K., Shibata Darryl, Schwarz Klaus, Hsieh Chih-Lin, Lieber Michael R. The embryonic lethality in DNA ligase IV-deficient mice is rescued by deletion of Ku: implications for unifying the heterogeneous phenotypes of NHEJ mutants. DNA Repair (Amst) 2002 Dec 5;1(12):1017–1026. doi: 10.1016/s1568-7864(02)00151-9. [DOI] [PubMed] [Google Scholar]
  31. Kooistra R., Pastink A., Zonneveld J. B., Lohman P. H., Eeken J. C. The Drosophila melanogaster DmRAD54 gene plays a crucial role in double-strand break repair after P-element excision and acts synergistically with Ku70 in the repair of X-ray damage. Mol Cell Biol. 1999 Sep;19(9):6269–6275. doi: 10.1128/mcb.19.9.6269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kooistra R., Vreeken K., Zonneveld J. B., de Jong A., Eeken J. C., Osgood C. J., Buerstedde J. M., Lohman P. H., Pastink A. The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination. Mol Cell Biol. 1997 Oct;17(10):6097–6104. doi: 10.1128/mcb.17.10.6097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ma Yunmei, Pannicke Ulrich, Schwarz Klaus, Lieber Michael R. Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell. 2002 Mar 22;108(6):781–794. doi: 10.1016/s0092-8674(02)00671-2. [DOI] [PubMed] [Google Scholar]
  34. Martin Ina V., MacNeill Stuart A. ATP-dependent DNA ligases. Genome Biol. 2002 Mar 19;3(4):REVIEWS3005–REVIEWS3005. doi: 10.1186/gb-2002-3-4-reviews3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Moshous D., Callebaut I., de Chasseval R., Corneo B., Cavazzana-Calvo M., Le Deist F., Tezcan I., Sanal O., Bertrand Y., Philippe N. Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell. 2001 Apr 20;105(2):177–186. doi: 10.1016/s0092-8674(01)00309-9. [DOI] [PubMed] [Google Scholar]
  36. Nick McElhinny S. A., Snowden C. M., McCarville J., Ramsden D. A. Ku recruits the XRCC4-ligase IV complex to DNA ends. Mol Cell Biol. 2000 May;20(9):2996–3003. doi: 10.1128/mcb.20.9.2996-3003.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nussenzweig A., Chen C., da Costa Soares V., Sanchez M., Sokol K., Nussenzweig M. C., Li G. C. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature. 1996 Aug 8;382(6591):551–555. doi: 10.1038/382551a0. [DOI] [PubMed] [Google Scholar]
  38. Pastink A., Eeken J. C., Lohman P. H. Genomic integrity and the repair of double-strand DNA breaks. Mutat Res. 2001 Sep 1;480-481:37–50. doi: 10.1016/s0027-5107(01)00167-1. [DOI] [PubMed] [Google Scholar]
  39. Richardson C., Jasin M. Coupled homologous and nonhomologous repair of a double-strand break preserves genomic integrity in mammalian cells. Mol Cell Biol. 2000 Dec;20(23):9068–9075. doi: 10.1128/mcb.20.23.9068-9075.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rong Y. S., Golic K. G. Gene targeting by homologous recombination in Drosophila. Science. 2000 Jun 16;288(5473):2013–2018. doi: 10.1126/science.288.5473.2013. [DOI] [PubMed] [Google Scholar]
  41. Sibanda B. L., Critchlow S. E., Begun J., Pei X. Y., Jackson S. P., Blundell T. L., Pellegrini L. Crystal structure of an Xrcc4-DNA ligase IV complex. Nat Struct Biol. 2001 Dec;8(12):1015–1019. doi: 10.1038/nsb725. [DOI] [PubMed] [Google Scholar]
  42. Siede W., Friedl A. A., Dianova I., Eckardt-Schupp F., Friedberg E. C. The Saccharomyces cerevisiae Ku autoantigen homologue affects radiosensitivity only in the absence of homologous recombination. Genetics. 1996 Jan;142(1):91–102. doi: 10.1093/genetics/142.1.91. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taccioli G. E., Amatucci A. G., Beamish H. J., Gell D., Xiang X. H., Torres Arzayus M. I., Priestley A., Jackson S. P., Marshak Rothstein A., Jeggo P. A. Targeted disruption of the catalytic subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity. Immunity. 1998 Sep;9(3):355–366. doi: 10.1016/s1074-7613(00)80618-4. [DOI] [PubMed] [Google Scholar]
  44. Van Dyck E., Stasiak A. Z., Stasiak A., West S. C. Binding of double-strand breaks in DNA by human Rad52 protein. Nature. 1999 Apr 22;398(6729):728–731. doi: 10.1038/19560. [DOI] [PubMed] [Google Scholar]
  45. Verkaik Nicole S., Esveldt-van Lange Rebecca E. E., van Heemst Diana, Brüggenwirth Hennie T., Hoeijmakers Jan H. J., Zdzienicka Malgorzata Z., van Gent Dik C. Different types of V(D)J recombination and end-joining defects in DNA double-strand break repair mutant mammalian cells. Eur J Immunol. 2002 Mar;32(3):701–709. doi: 10.1002/1521-4141(200203)32:3<701::AID-IMMU701>3.0.CO;2-T. [DOI] [PubMed] [Google Scholar]
  46. Wei Y. F., Robins P., Carter K., Caldecott K., Pappin D. J., Yu G. L., Wang R. P., Shell B. K., Nash R. A., Schär P. 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. 1995 Jun;15(6):3206–3216. doi: 10.1128/mcb.15.6.3206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zhu C., Bogue M. A., Lim D. S., Hasty P., Roth D. B. Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell. 1996 Aug 9;86(3):379–389. doi: 10.1016/s0092-8674(00)80111-7. [DOI] [PubMed] [Google Scholar]

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