Plant Molecular Biology

, Volume 50, Issue 1, pp 69–79 | Cite as

Molecular cloning and characterization of RAD51-like genes from Arabidopsis thaliana

  • Keishi Osakabe
  • Toji Yoshioka
  • Hiroaki Ichikawa
  • Seiichi Toki


Homologous recombination is an essential process for the maintenance and variability of the genome. In eukaryotes, the Rad52 epistasis group proteins serve the main role for meiotic recombination and/or homologous recombinational repair. Rad51-like proteins, such as Rad55 and Rad57 in yeast, play a role in assembly or stabilization of multimeric Rad51 that promotes homologous pairing and strand exchange reactions. We cloned two RAD51-like genes named AtXRCC3 and AtRAD51C from Arabidopsis thaliana. Both AtXRCC3 and AtRAD51C expressed two alternatively spliced transcripts, and AtRAD51C produced two different sizes of isoforms, a long (AtRAD51Cα) and a short one (AtRAD51Cβ). The predicted protein sequences of these genes showed characteristic features of the RecA/Rad51 family; especially the amino acids around the ATP-binding motifs were well conserved. The transcripts of AtXRCC3 and AtRAD51C were detected in various tissues, with the highest level of expression in flower buds. Expression of both genes was induced by γ-ray irradiation. The results of yeast two-hybrid assays suggested that Arabidopsis Rad51 family proteins form a complex, which could participate in meiotic recombination and/or homologous recombinational repair.

Arabidopsis homologous recombination Rad51C Rad51-like genes Xrcc3 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ahmad, M., Jarillo, J.A., Kilmczak, L.J., Landy, L.G., Pag, T., Last, R.L. and Cashmore, A.R. 1997. An enzyme similar to animal type II photolyases mediates photoreactivation in Arabidopsis. Plant Cell 9: 199–207.Google Scholar
  2. Aihara, H., Ito, Y., Kurumizaka, H., Yokoyama, S. and Shibata, T. 1999. The N-terminal domain of the human Rad51 protein binds DNA: Structure and a DNA binding surface as revealed by NMR. J. Mol. Biol. 290: 495–504.Google Scholar
  3. Arabidopsis Initiative 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796–815.Google Scholar
  4. Baumann, P. and West, S.C. 1998. Role of human Rad51 protein in homologous recombination and double-stranded-break repair. Trends Biochem. Sci. 23: 247–251.Google Scholar
  5. Bishop, D.K., Park, D., Xu, L. and Kleckner, N. 1992. DMC1: A meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 69: 439–456.Google Scholar
  6. Bishop, D.K., Ear, U., Bhattacharyya, A., Calderone, C., Beckett, M., Weichselbaum, R.R. and Shinohara, A. 1998. Xrcc3 is required for assembly of Rad51 complexs in vivo. J. Biol. Chem. 273: 21482–21488.Google Scholar
  7. Cartwright, R., Dunn, A.M., Simpson, P.J., Tambini, C.E. and Thacker, J. 1998. Isolation of novel human and mouse genes of the recA/RAD51 recombination-repair gene family. Nucl. Acids. Res. 26: 165–1659.Google Scholar
  8. Couteau, F., Belzile, F., Horlow, C., Grandjean, O., Vezon, D. and Doutriaux, M.P. 1999. Random chromosome segregation without meiotic arrest in both male and female meiocytes of a dmc1 mutant of Arabidopsis. Plant Cell 11: 1623–1634.Google Scholar
  9. Donovan, J.W., Milne, G.T. and Weaver, D.T. 1994. Homotypic and heterotypic protein associations control rad51 function in double-strand break repair. Genes Dev. 8: 2552–2562.Google Scholar
  10. Dosanjh, M.K., Collins, D.W., Fan, W., Lennon, G., Albana, J.S., Shen, Z. and Schild, D. 1998. Isolation and characterization of RAD51C, a new human member of the RAD51 family of related genes. Nucl. Acids. Res. 26: 1179–1184.Google Scholar
  11. Doutriaux, M.-P., Couteau, F., Bergounioux, C. and White, C. 1998. Isolation and characterization of the RAD51 and DMC1 homologs from Arabidopsis thaliana. Mol. Gen. Genet. 257: 283–291.Google Scholar
  12. Fuller, L.F. and Painter, R.B. 1988. A Chinese hamster ovary cell line hypersensitive to ionizing radiation and deficient in repair replication. Mutat. Res. 193: 109–121.Google Scholar
  13. Gallego, F., Fleck, O., Li, A., Wyrzykowsky, J. and Tinland, B. 2000. AtRAD1, a plant homologue of human and yeast nucleotide excision repair endonucleases, is involved in dark repair of UV damages and recombination. Plant J. 21: 507–518.Google Scholar
  14. Gallego, M.E., Jeanneau, M., Granier, F., Bouchez, D., Bechtold, N. and White, C.I. 2001. Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity. Plant J. 25: 31–41.Google Scholar
  15. Gorbunova, V. and Levy, A.A. 1999. How plants make ends meet: DNA double-strand break repair. Trends Plant Sci. 4: 263–269.Google Scholar
  16. Gupta, R.C., Bazemore, L.R., Golub, E.I. and Radding, C.M. 1997. Activities of human recombination protein Rad51. Proc. Natl. Acad. Sci. USA 94: 463–468.Google Scholar
  17. Habu, T., Taki, T., West, A., Nishimune, Y. and Morita, T. 1996. The mouse and human homologs of DMC1, the yeast meiosis-specific homologous recombination gene, have a common unique form of exon-skipped transcript in meiosis. Nucl. Acids. Res. 24: 470–477.Google Scholar
  18. Hays, S.L., Firmenich, A.A. and Berg, P. 1995. Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55 and Rad57 proteins. Proc. Natl. Acad. Sci. USA 92: 6925–6929.Google Scholar
  19. Higgins, C.F., Hiles, I.D., Whalley, K. and Jamieson, D.J. 1985. Nucleotide binding by membrane components of bacterial periplasmic binding protein-dependent transport systems. EMBO J. 4: 103–1039.Google Scholar
  20. Jang, Y.K., Jin, Y.H., Shim, Y.S., Kim, M.J., Yoo, E.J., Choi, L.S., Lee, S.J., Seong, R.H., Hong, S.H. and Park, S.D. 1996. Identification of the DNA damage-responsive elements of the rhp51+ gene, a recA and RAD51 homolog from the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 251: 167–175.Google Scholar
  21. Jiang, H., Xie, Y., Houston, P., Stemke-Hale, K., Mortensen, U.H., Rothstein, R. and Kodadek, T. 1996. Direct association between the yeast Rad51 and Rad54 recombination proteins. J. Biol. Chem. 271: 33181–33186.Google Scholar
  22. Johnson, R.D. and Symington, L.S. 1995. Functional differences and interactions among the putative RecA homologs Rad51, Rad55, and Rad57. Mol. Cello Biol. 15: 4843–4850.Google Scholar
  23. Johnson, R.D., Liu, N. and Jasin, M. 1999. Mammalian XRCC2 promotes the repair of DNA double-strand breaks by homologous recombination. Nature 401: 397–399.Google Scholar
  24. Jones, N.J., Cox, R. and Thacker, J. 1987. Isolation and cross-sensitivity of X-ray-sensitivity mutants of V79-4 hamster cells. Mutat. Res. 183: 279–286.Google Scholar
  25. Kans, J.A. and Mortimer, R.K. 1991. Nucleotide sequence of the RAD57 gene of Saccharomyces cerevisiae. Gene 105: 139–140.Google Scholar
  26. Kawabata, M. and Saeki, K. 1999. Multiple alternative transcripts of the human homologue of the mouse TRAD/R51H3/RAD51D gene, a member of the recA/RAD51 gene family. Biochem. Biophys. Res. Commun. 257: 156–162.Google Scholar
  27. Kempin, S., Liljegren, S.J., Block, L.M., Rounsley, S.D., Yanofsky, M.F. and Lam, E. 1997. Targeted disruption in Arabidopsis. Nature 389: 802–803.Google Scholar
  28. Klimyuk, V.I. and Jones, J.D.G. 1997. AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression. Plant J. 11: 1–14.Google Scholar
  29. Krejci, L., Damborsky, J., Thomsen, B., Duno, M. and Bendixen, C. 2001. Molecular dissection of interactions between Rad51 and members of the recombination-repair group. Mol. Cell Biol. 21: 966–976.Google Scholar
  30. Lee, K. Y., Lund, P., Lowe, K. and Dunsmuir, P. 1990. Homologous recombination in plant cells after Agrobacterium-mediated transformation. Plant Cell 4: 415–425.Google Scholar
  31. Lovett, S.T. 1994. Sequence of the RAD55 gene of Saccharomyces cerevisiae: similar to prokaryotic RecA and other RecA-like proteins. Gene 142: 103–106.Google Scholar
  32. Lovett, S.T. and Mortimer, R.K. 1987. Characterization of null mutants of RAD55 gene of Saccharomyces cerevisiae: effects of temperature, osmotic strength and mating type. Genetics 116: 547–553.Google Scholar
  33. Masson, J.-Y., Stasiak, A.Z., Stasiak, A., Benson, F. and West, S.C. 2001. Complex formation by the human RAD51C and XRCC3 recombination repair proteins. Proc. Natl. Acad. Sci. USA 98: 8440–8446.Google Scholar
  34. Mengiste, T. and Paszkowski, J. 1999. Prospects for the precise engineering of plant genomes by homologous recombination. Biol. Chem. 380: 749–758.Google Scholar
  35. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15: 473–497.Google Scholar
  36. Offringa, R., de Groot, J.A., Haagsman, H.J., Does, M.P., van den Elzen, P.J.M. and Hooykaas, P.J.J. 1990. Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium mediated transformation. EMBO J. 9: 3077–3084.Google Scholar
  37. Ogawa, T., Yu, X., Shinohara, A. and Egleman, E.H. 1993. Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science 259: 1896–1899.Google Scholar
  38. Page, R.D.M. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Comp. Appli. Biosci. 12: 357–358.Google Scholar
  39. Passy, S.I., yu, X., Li, Z., Radding, ZC.M., Masson, J.-Y., West, S.C. and Egelman, E.H. 1999. Human Dmc1 protein binds DNA as an octameric ring. Proc. Natl. Acad. Sci. USA 96: 10684–10688.Google Scholar
  40. Paszkowski, J., Baur, M., Bogucki, A. and Potrykus, I. 1988. Gene targeting in plants. EMBO J. 7: 4021–4026.Google Scholar
  41. Peirson, B.N., Owen, H.A., Feldmann, K.A. and Makaroff, C.A. 1996. Characterization of three male-sterile mutants of Arabidopsis thaliana exhibiting alterations in meiosis. Sex. Plant Reprod. 9: 1–16.Google Scholar
  42. Pittman, D.L., Weinberg, L.R. and Schimenti, J.C. 1998. Identification, characterization, and genetic mapping of Rad51d, a new mouse and human RAD51/RecA-related gene. Genomics 49: 103–111.Google Scholar
  43. Puchta, H. and Hohn, B. 1996. From centiMorgans to base pairs: homologous recombination in plants. Trends Plant Sci. 1: 340–348.Google Scholar
  44. Rice, M.C., Smith, S.T., Bullrich, F., Havre, P. and Kmiec, E.B. 1997. Isolation of human and mouse genes based on homology to REC2, a recombinational repair gene from the fungus Ustilago maydis. Proc. Natl. Acad. Sci. USA 94: 7417–7422.Google Scholar
  45. Schild, D., Lio, Y.-C., Collins, D.W., Tsomondo, T. and Chen, D.J. 2000. Evidence for simultaneous protein interactions between human RAD51 paralogs. J. Biol. Chem. 275: 16443–16449.Google Scholar
  46. Shen, Z, Cloud, K.G, Chen, D.J. and Park, M.S. 1996. Specific interactions between the human RAD51 and RAD52 proteins. J. Biol. Chem. 271: 148–152.Google Scholar
  47. Shinohara, A. and Ogawa, T. 1995. Homologous recombination and the roles of double-strand breaks. Trends Biochem. Sci. 20: 387–391.Google Scholar
  48. Shu, Z., Smith, S., Wang, L., Rice, M.C. and Kmiec, E.B. 1999. Disruption of muREC2/RAD51L1 in mice results in early embryonic lethality which can be partially rescued in a p53 ?/? background. Mol. Cell. Biol. 19: 8686–8693.Google Scholar
  49. Sung, P. 1994. Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast Rad51 protein. Science 278: 1241–1243.Google Scholar
  50. Sung, P. 1997. Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev. 11: 1111–1121.Google Scholar
  51. Takata, M., Sasaki, M.S., Sonoda, E., Fukushima, T., Morrison, C., Albala, J., Swagemakers, M.A., Kanaar, R., Thompson, L.H. and Takeda, S. 2000. The Rad51 paralogs Rad51B promotes homologous recombinational repair. Mol. Cell. Biol. 20: 6476–6482.Google Scholar
  52. Tebbs, R.S., Zhao, Y., Tucker, J.D., Scheerer, J.B., Siciliano, M.J., Hwang, M., Liu, N., Legerski, R.J. and Thompson, L.H. 1995. Correction of chromosomal instability and sensitivity to diverse mutagens by cDNA of the XRCC3 DNA repair gene. Proc. Natl. Acad. Sci. USA 92: 6354–6358.Google Scholar
  53. Thacker, J., Tambini, C.E., Simpson, P.J., Tsui, L.C. and Scherer, S.W. 1995. Localization of chromosome 7q36.1 of the human XRCC2 gene, determining sensitivity to DNA-damaging agents. Hum. Mol. Genet. 4: 113–120.Google Scholar
  54. Thompson, J.D., Higgins, D.G. and Gibson, T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22: 4673–4680.Google Scholar
  55. Tsuzuki, T., Fujii, Y., Sakuma, K., Tominaga, Y., Nakano, K., Sekiguchi, M., Matsushiro, A., Yoshimura, Y. and Morita, T. 1996. Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc. Natl. Acad. Sci. USA 93: 6236–6240.Google Scholar
  56. Trujillo, K.M., Yuan, S.S., Lee, E.Y. and Sung, P. 1998. Nuclease activities in a complex of human recombination and DNA repair factors Rad50, Mre11 and p95. J. Biol. Chem. 273: 21447–21450.Google Scholar
  57. Walker, J.E., Saraste, M., Runswick, M.J. and Gay, N.J. 1982. Distantly related sequences in the a-and b-subunits of ATP-synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1: 945–951.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Keishi Osakabe
    • 1
  • Toji Yoshioka
    • 2
  • Hiroaki Ichikawa
    • 1
  • Seiichi Toki
    • 1
  1. 1.Department of Plant BiotechnologyNational Institute of Agrobiological SciencesTsukuba, IbarakiJapan
  2. 2.Headquarters of National Agricultural Research OrganizationTsukuba, IbarakiJapan

Personalised recommendations