Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Oct;17(10):6097–6104. doi: 10.1128/mcb.17.10.6097

The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination.

R Kooistra 1, K Vreeken 1, J B Zonneveld 1, A de Jong 1, J C Eeken 1, C J Osgood 1, J M Buerstedde 1, P H Lohman 1, A Pastink 1
PMCID: PMC232459  PMID: 9315669

Abstract

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRADS4-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aboussekhra A., Chanet R., Adjiri A., Fabre F. Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. Mol Cell Biol. 1992 Jul;12(7):3224–3234. doi: 10.1128/mcb.12.7.3224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akaboshi E., Inoue Y., Ryo H. Cloning of the cDNA and genomic DNA that correspond to the recA-like gene of Drosophila melanogaster. Jpn J Genet. 1994 Dec;69(6):663–670. doi: 10.1266/jjg.69.663. [DOI] [PubMed] [Google Scholar]
  3. Bai Y., Symington L. S. A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae. Genes Dev. 1996 Aug 15;10(16):2025–2037. doi: 10.1101/gad.10.16.2025. [DOI] [PubMed] [Google Scholar]
  4. Basile G., Aker M., Mortimer R. K. Nucleotide sequence and transcriptional regulation of the yeast recombinational repair gene RAD51. Mol Cell Biol. 1992 Jul;12(7):3235–3246. doi: 10.1128/mcb.12.7.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beall E. L., Rio D. C. Drosophila IRBP/Ku p70 corresponds to the mutagen-sensitive mus309 gene and is involved in P-element excision in vivo. Genes Dev. 1996 Apr 15;10(8):921–933. doi: 10.1101/gad.10.8.921. [DOI] [PubMed] [Google Scholar]
  6. Bendixen C., Sunjevaric I., Bauchwitz R., Rothstein R. Identification of a mouse homologue of the Saccharomyces cerevisiae recombination and repair gene, RAD52. Genomics. 1994 Sep 1;23(1):300–303. doi: 10.1006/geno.1994.1503. [DOI] [PubMed] [Google Scholar]
  7. Bezzubova O. Y., Schmidt H., Ostermann K., Heyer W. D., Buerstedde J. M. Identification of a chicken RAD52 homologue suggests conservation of the RAD52 recombination pathway throughout the evolution of higher eukaryotes. Nucleic Acids Res. 1993 Dec 25;21(25):5945–5949. doi: 10.1093/nar/21.25.5945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bezzubova O., Shinohara A., Mueller R. G., Ogawa H., Buerstedde J. M. A chicken RAD51 homologue is expressed at high levels in lymphoid and reproductive organs. Nucleic Acids Res. 1993 Apr 11;21(7):1577–1580. doi: 10.1093/nar/21.7.1577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Cavener D. R. Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 1987 Feb 25;15(4):1353–1361. doi: 10.1093/nar/15.4.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chepurnaya O. V., Kozhin S. A., Peshekhonov V. T., Korolev V. G. RAD58 (XRS4)--a new gene in the RAD52 epistasis group. Curr Genet. 1995 Aug;28(3):274–279. doi: 10.1007/BF00309787. [DOI] [PubMed] [Google Scholar]
  12. Contopoulou C. R., Cook V. E., Mortimer R. K. Analysis of DNA double strand breakage and repair using orthogonal field alternation gel electrophoresis. Yeast. 1987 Jun;3(2):71–76. doi: 10.1002/yea.320030203. [DOI] [PubMed] [Google Scholar]
  13. Dolganov G. M., Maser R. S., Novikov A., Tosto L., Chong S., Bressan D. A., Petrini J. H. Human Rad50 is physically associated with human Mre11: identification of a conserved multiprotein complex implicated in recombinational DNA repair. Mol Cell Biol. 1996 Sep;16(9):4832–4841. doi: 10.1128/mcb.16.9.4832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eisen J. A., Sweder K. S., Hanawalt P. C. Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions. Nucleic Acids Res. 1995 Jul 25;23(14):2715–2723. doi: 10.1093/nar/23.14.2715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Flygare J., Benson F., Hellgren D. Expression of the human RAD51 gene during the cell cycle in primary human peripheral blood lymphocytes. Biochim Biophys Acta. 1996 Jul 24;1312(3):231–236. doi: 10.1016/0167-4889(96)00040-7. [DOI] [PubMed] [Google Scholar]
  16. Game J. C. DNA double-strand breaks and the RAD50-RAD57 genes in Saccharomyces. Semin Cancer Biol. 1993 Apr;4(2):73–83. [PubMed] [Google Scholar]
  17. Godwin A. R., Bollag R. J., Christie D. M., Liskay R. M. Spontaneous and restriction enzyme-induced chromosomal recombination in mammalian cells. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12554–12558. doi: 10.1073/pnas.91.26.12554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jacoby D. B., Wensink P. C. Yolk protein factor 1 is a Drosophila homolog of Ku, the DNA-binding subunit of a DNA-dependent protein kinase from humans. J Biol Chem. 1994 Apr 15;269(15):11484–11491. [PubMed] [Google Scholar]
  19. Jang Y. K., Jin Y. H., Kim E. M., Fabre F., Hong S. H., Park S. D. Cloning and sequence analysis of rhp51+, a Schizosaccharomyces pombe homolog of the Saccharomyces cerevisiae RAD51 gene. Gene. 1994 May 16;142(2):207–211. doi: 10.1016/0378-1119(94)90262-3. [DOI] [PubMed] [Google Scholar]
  20. Jeggo P. A., Taccioli G. E., Jackson S. P. Menage à trois: double strand break repair, V(D)J recombination and DNA-PK. Bioessays. 1995 Nov;17(11):949–957. doi: 10.1002/bies.950171108. [DOI] [PubMed] [Google Scholar]
  21. Jeong-Yu S. J., Carroll D. Test of the double-strand-break repair model of recombination in Xenopus laevis oocytes. Mol Cell Biol. 1992 Jan;12(1):112–119. doi: 10.1128/mcb.12.1.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kanaar R., Troelstra C., Swagemakers S. M., Essers J., Smit B., Franssen J. H., Pastink A., Bezzubova O. Y., Buerstedde J. M., Clever B. Human and mouse homologs of the Saccharomyces cerevisiae RAD54 DNA repair gene: evidence for functional conservation. Curr Biol. 1996 Jul 1;6(7):828–838. doi: 10.1016/s0960-9822(02)00606-1. [DOI] [PubMed] [Google Scholar]
  23. Lehman C. W., Trautman J. K., Carroll D. Illegitimate recombination in Xenopus: characterization of end-joined junctions. Nucleic Acids Res. 1994 Feb 11;22(3):434–442. doi: 10.1093/nar/22.3.434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lim D. S., Hasty P. A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol Cell Biol. 1996 Dec;16(12):7133–7143. doi: 10.1128/mcb.16.12.7133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lin F. L., Sperle K., Sternberg N. Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells. Mol Cell Biol. 1990 Jan;10(1):113–119. doi: 10.1128/mcb.10.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lukacsovich T., Yang D., Waldman A. S. Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI. Nucleic Acids Res. 1994 Dec 25;22(25):5649–5657. doi: 10.1093/nar/22.25.5649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McKee B. D., Ren X., Hong C. A recA-like gene in Drosophila melanogaster that is expressed at high levels in female but not male meiotic tissues. Chromosoma. 1996 Apr;104(7):479–488. doi: 10.1007/BF00352112. [DOI] [PubMed] [Google Scholar]
  28. Milne G. T., Jin S., Shannon K. B., Weaver D. T. Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Aug;16(8):4189–4198. doi: 10.1128/mcb.16.8.4189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Morita T., Yoshimura Y., Yamamoto A., Murata K., Mori M., Yamamoto H., Matsushiro A. A mouse homolog of the Escherichia coli recA and Saccharomyces cerevisiae RAD51 genes. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6577–6580. doi: 10.1073/pnas.90.14.6577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mortensen U. H., Bendixen C., Sunjevaric I., Rothstein R. DNA strand annealing is promoted by the yeast Rad52 protein. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10729–10734. doi: 10.1073/pnas.93.20.10729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mozer B., Marlor R., Parkhurst S., Corces V. Characterization and developmental expression of a Drosophila ras oncogene. Mol Cell Biol. 1985 Apr;5(4):885–889. doi: 10.1128/mcb.5.4.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Muris D. F., Bezzubova O., Buerstedde J. M., Vreeken K., Balajee A. S., Osgood C. J., Troelstra C., Hoeijmakers J. H., Ostermann K., Schmidt H. Cloning of human and mouse genes homologous to RAD52, a yeast gene involved in DNA repair and recombination. Mutat Res. 1994 Nov;315(3):295–305. doi: 10.1016/0921-8777(94)90040-x. [DOI] [PubMed] [Google Scholar]
  33. Muris D. F., Vreeken K., Carr A. M., Broughton B. C., Lehmann A. R., Lohman P. H., Pastink A. Cloning the RAD51 homologue of Schizosaccharomyces pombe. Nucleic Acids Res. 1993 Sep 25;21(19):4586–4591. doi: 10.1093/nar/21.19.4586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Muris D. F., Vreeken K., Carr A. M., Murray J. M., Smit C., Lohman P. H., Pastink A. Isolation of the Schizosaccharomyces pombe RAD54 homologue, rhp54+, a gene involved in the repair of radiation damage and replication fidelity. J Cell Sci. 1996 Jan;109(Pt 1):73–81. doi: 10.1242/jcs.109.1.73. [DOI] [PubMed] [Google Scholar]
  35. Ostermann K., Lorentz A., Schmidt H. The fission yeast rad22 gene, having a function in mating-type switching and repair of DNA damages, encodes a protein homolog to Rad52 of Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Dec 25;21(25):5940–5944. doi: 10.1093/nar/21.25.5940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pastink A., Heemskerk E., Nivard M. J., van Vliet C. J., Vogel E. W. Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster. Mol Gen Genet. 1991 Oct;229(2):213–218. doi: 10.1007/BF00272158. [DOI] [PubMed] [Google Scholar]
  37. Petrini J. H., Walsh M. E., DiMare C., Chen X. N., Korenberg J. R., Weaver D. T. Isolation and characterization of the human MRE11 homologue. Genomics. 1995 Sep 1;29(1):80–86. doi: 10.1006/geno.1995.1217. [DOI] [PubMed] [Google Scholar]
  38. Price A. The repair of ionising radiation-induced damage to DNA. Semin Cancer Biol. 1993 Apr;4(2):61–71. [PubMed] [Google Scholar]
  39. Roth D. B., Lindahl T., Gellert M. Repair and recombination. How to make ends meet. Curr Biol. 1995 May 1;5(5):496–499. doi: 10.1016/s0960-9822(95)00101-1. [DOI] [PubMed] [Google Scholar]
  40. Sander M., Lowenhaupt K., Lane W. S., Rich A. Cloning and characterization of Rrp1, the gene encoding Drosophila strand transferase: carboxy-terminal homology to DNA repair endo/exonucleases. Nucleic Acids Res. 1991 Aug 25;19(16):4523–4529. doi: 10.1093/nar/19.16.4523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Shen Z., Denison K., Lobb R., Gatewood J. M., Chen D. J. The human and mouse homologs of the yeast RAD52 gene: cDNA cloning, sequence analysis, assignment to human chromosome 12p12.2-p13, and mRNA expression in mouse tissues. Genomics. 1995 Jan 1;25(1):199–206. doi: 10.1016/0888-7543(95)80126-7. [DOI] [PubMed] [Google Scholar]
  43. Shinohara A., Ogawa H., Matsuda Y., Ushio N., Ikeo K., Ogawa T. Cloning of human, mouse and fission yeast recombination genes homologous to RAD51 and recA. Nat Genet. 1993 Jul;4(3):239–243. doi: 10.1038/ng0793-239. [DOI] [PubMed] [Google Scholar]
  44. Shinohara A., Ogawa H., Ogawa T. Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell. 1992 May 1;69(3):457–470. doi: 10.1016/0092-8674(92)90447-k. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Sung P., Robberson D. L. DNA strand exchange mediated by a RAD51-ssDNA nucleoprotein filament with polarity opposite to that of RecA. Cell. 1995 Aug 11;82(3):453–461. doi: 10.1016/0092-8674(95)90434-4. [DOI] [PubMed] [Google Scholar]
  47. Taki T., Ohnishi T., Yamamoto A., Hiraga S., Arita N., Izumoto S., Hayakawa T., Morita T. Antisense inhibition of the RAD51 enhances radiosensitivity. Biochem Biophys Res Commun. 1996 Jun 14;223(2):434–438. doi: 10.1006/bbrc.1996.0911. [DOI] [PubMed] [Google Scholar]
  48. Tashiro S., Kotomura N., Shinohara A., Tanaka K., Ueda K., Kamada N. S phase specific formation of the human Rad51 protein nuclear foci in lymphocytes. Oncogene. 1996 May 16;12(10):2165–2170. [PubMed] [Google Scholar]
  49. Tavassoli M., Shayeghi M., Nasim A., Watts F. Z. Cloning and characterisation of the Schizosaccharomyces pombe rad32 gene: a gene required for repair of double strand breaks and recombination. Nucleic Acids Res. 1995 Feb 11;23(3):383–388. doi: 10.1093/nar/23.3.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Troelstra C., van Gool A., de Wit J., Vermeulen W., Bootsma D., Hoeijmakers J. H. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes. Cell. 1992 Dec 11;71(6):939–953. doi: 10.1016/0092-8674(92)90390-x. [DOI] [PubMed] [Google Scholar]
  51. Tsuzuki T., Fujii Y., Sakumi K., Tominaga Y., Nakao K., Sekiguchi M., Matsushiro A., Yoshimura Y., MoritaT Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6236–6240. doi: 10.1073/pnas.93.13.6236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vogel E. W. Tests for recombinagens in somatic cells of Drosophila. Mutat Res. 1992 Dec 1;284(1):159–175. doi: 10.1016/0027-5107(92)90030-6. [DOI] [PubMed] [Google Scholar]
  53. Weaver D. T. What to do at an end: DNA double-strand-break repair. Trends Genet. 1995 Oct;11(10):388–392. doi: 10.1016/s0168-9525(00)89121-0. [DOI] [PubMed] [Google Scholar]
  54. Yamamoto A., Taki T., Yagi H., Habu T., Yoshida K., Yoshimura Y., Yamamoto K., Matsushiro A., Nishimune Y., Morita T. Cell cycle-dependent expression of the mouse Rad51 gene in proliferating cells. Mol Gen Genet. 1996 Apr 24;251(1):1–12. doi: 10.1007/BF02174338. [DOI] [PubMed] [Google Scholar]
  55. Zdzienicka M. Z. Mammalian mutants defective in the response to ionizing radiation-induced DNA damage. Mutat Res. 1995 May;336(3):203–213. doi: 10.1016/0921-8777(95)00003-3. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES