Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 Jul 1;26(13):3165–3172. doi: 10.1093/nar/26.13.3165

Identification and characterization of the RAD51 gene from the ciliate Tetrahymena thermophila.

C Campbell 1, D P Romero 1
PMCID: PMC147671  PMID: 9628914

Abstract

The RAD51 gene is a eukaryotic homolog of rec A, a critical component in homologous recombination and DNA repair pathways in Escherichia coli . We have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single- and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.

Full Text

The Full Text of this article is available as a PDF (228.2 KB).

Selected References

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

  1. 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]
  2. Baumann P., Benson F. E., West S. C. Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell. 1996 Nov 15;87(4):757–766. doi: 10.1016/s0092-8674(00)81394-x. [DOI] [PubMed] [Google Scholar]
  3. Benson F. E., Stasiak A., West S. C. Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J. 1994 Dec 1;13(23):5764–5771. doi: 10.1002/j.1460-2075.1994.tb06914.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Brendel V., Brocchieri L., Sandler S. J., Clark A. J., Karlin S. Evolutionary comparisons of RecA-like proteins across all major kingdoms of living organisms. J Mol Evol. 1997 May;44(5):528–541. doi: 10.1007/pl00006177. [DOI] [PubMed] [Google Scholar]
  6. Buchhop S., Gibson M. K., Wang X. W., Wagner P., Stürzbecher H. W., Harris C. C. Interaction of p53 with the human Rad51 protein. Nucleic Acids Res. 1997 Oct 1;25(19):3868–3874. doi: 10.1093/nar/25.19.3868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Butler D. K., Yasuda L. E., Yao M. C. Induction of large DNA palindrome formation in yeast: implications for gene amplification and genome stability in eukaryotes. Cell. 1996 Dec 13;87(6):1115–1122. doi: 10.1016/s0092-8674(00)81805-x. [DOI] [PubMed] [Google Scholar]
  8. Camerini-Otero R. D., Hsieh P. Homologous recombination proteins in prokaryotes and eukaryotes. Annu Rev Genet. 1995;29:509–552. doi: 10.1146/annurev.ge.29.120195.002453. [DOI] [PubMed] [Google Scholar]
  9. Collins K., Greider C. W. Utilization of ribonucleotides and RNA primers by Tetrahymena telomerase. EMBO J. 1995 Nov 1;14(21):5422–5432. doi: 10.1002/j.1460-2075.1995.tb00226.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cox M. M. Relating biochemistry to biology: how the recombinational repair function of RecA protein is manifested in its molecular properties. Bioessays. 1993 Sep;15(9):617–623. doi: 10.1002/bies.950150908. [DOI] [PubMed] [Google Scholar]
  11. Coyne R. S., Chalker D. L., Yao M. C. Genome downsizing during ciliate development: nuclear division of labor through chromosome restructuring. Annu Rev Genet. 1996;30:557–578. doi: 10.1146/annurev.genet.30.1.557. [DOI] [PubMed] [Google Scholar]
  12. Coyne R. S., Yao M. C. Evolutionary conservation of sequences directing chromosome breakage and rDNA palindrome formation in tetrahymenine ciliates. Genetics. 1996 Dec;144(4):1479–1487. doi: 10.1093/genetics/144.4.1479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gassmann M., Thömmes P., Weiser T., Hübscher U. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J. 1990 May;4(8):2528–2532. doi: 10.1096/fasebj.4.8.1970792. [DOI] [PubMed] [Google Scholar]
  14. Haaf T., Golub E. I., Reddy G., Radding C. M., Ward D. C. Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes. Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2298–2302. doi: 10.1073/pnas.92.6.2298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haddad A., Turkewitz A. P. Analysis of exocytosis mutants indicates close coupling between regulated secretion and transcription activation in Tetrahymena. Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10675–10680. doi: 10.1073/pnas.94.20.10675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Horowitz S., Gorovsky M. A. An unusual genetic code in nuclear genes of Tetrahymena. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2452–2455. doi: 10.1073/pnas.82.8.2452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Kapler G. M. Developmentally regulated processing and replication of the Tetrahymena rDNA minichromosome. Curr Opin Genet Dev. 1993 Oct;3(5):730–735. doi: 10.1016/s0959-437x(05)80091-7. [DOI] [PubMed] [Google Scholar]
  19. Labeit S., Lehrach H., Goody R. S. DNA sequencing using alpha-thiodeoxynucleotides. Methods Enzymol. 1987;155:166–177. doi: 10.1016/0076-6879(87)55015-7. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Li M. J., Peakman M. C., Golub E. I., Reddy G., Ward D. C., Radding C. M., Maizels N. Rad51 expression and localization in B cells carrying out class switch recombination. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10222–10227. doi: 10.1073/pnas.93.19.10222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Li Z., Golub E. I., Gupta R., Radding C. M. Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein. Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11221–11226. doi: 10.1073/pnas.94.21.11221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Love H. D., Jr, Allen-Nash A., Zhao Q. A., Bannon G. A. mRNA stability plays a major role in regulating the temperature-specific expression of a Tetrahymena thermophila surface protein. Mol Cell Biol. 1988 Jan;8(1):427–432. doi: 10.1128/mcb.8.1.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Martindale D. W. Codon usage in Tetrahymena and other ciliates. J Protozool. 1989 Jan-Feb;36(1):29–34. doi: 10.1111/j.1550-7408.1989.tb02679.x. [DOI] [PubMed] [Google Scholar]
  26. McCormick-Graham M., Haynes W. J., Romero D. P. Variable telomeric repeat synthesis in Paramecium tetraurelia is consistent with misincorporation by telomerase. EMBO J. 1997 Jun 2;16(11):3233–3242. doi: 10.1093/emboj/16.11.3233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McCormick-Graham M., Romero D. P. A single telomerase RNA is sufficient for the synthesis of variable telomeric DNA repeats in ciliates of the genus Paramecium. Mol Cell Biol. 1996 Apr;16(4):1871–1879. doi: 10.1128/mcb.16.4.1871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McCormick-Graham M., Romero D. P. Ciliate telomerase RNA structural features. Nucleic Acids Res. 1995 Apr 11;23(7):1091–1097. doi: 10.1093/nar/23.7.1091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ogawa T., Yu X., Shinohara A., Egelman E. H. Similarity of the yeast RAD51 filament to the bacterial RecA filament. Science. 1993 Mar 26;259(5103):1896–1899. doi: 10.1126/science.8456314. [DOI] [PubMed] [Google Scholar]
  30. Roca A. I., Cox M. M. The RecA protein: structure and function. Crit Rev Biochem Mol Biol. 1990;25(6):415–456. doi: 10.3109/10409239009090617. [DOI] [PubMed] [Google Scholar]
  31. Romero D. P., Arredondo J. A., Traut R. R. Identification of a region of Escherichia coli ribosomal protein L2 required for the assembly of L16 into the 50 S ribosomal subunit. J Biol Chem. 1990 Oct 25;265(30):18185–18191. [PubMed] [Google Scholar]
  32. Romero D. P., Blackburn E. H. A conserved secondary structure for telomerase RNA. Cell. 1991 Oct 18;67(2):343–353. doi: 10.1016/0092-8674(91)90186-3. [DOI] [PubMed] [Google Scholar]
  33. Rusche J. R., Konigsberg W., Howard-Flanders P. Isolation of altered recA polypeptides and interaction with ATP and DNA. J Biol Chem. 1985 Jan 25;260(2):949–955. [PubMed] [Google Scholar]
  34. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  35. Sandler S. J., Satin L. H., Samra H. S., Clark A. J. recA-like genes from three archaean species with putative protein products similar to Rad51 and Dmc1 proteins of the yeast Saccharomyces cerevisiae. Nucleic Acids Res. 1996 Jun 1;24(11):2125–2132. doi: 10.1093/nar/24.11.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Sogin M. L., Morrison H. G., Hinkle G., Silberman J. D. Ancestral relationships of the major eukaryotic lineages. Microbiologia. 1996 Mar;12(1):17–28. [PubMed] [Google Scholar]
  38. Stargell L. A., Gorovsky M. A. TATA-binding protein and nuclear differentiation in Tetrahymena thermophila. Mol Cell Biol. 1994 Jan;14(1):723–734. doi: 10.1128/mcb.14.1.723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stargell L. A., Karrer K. M., Gorovsky M. A. Transcriptional regulation of gene expression in Tetrahymena thermophila. Nucleic Acids Res. 1990 Nov 25;18(22):6637–6639. doi: 10.1093/nar/18.22.6637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stassen N. Y., Logsdon J. M., Jr, Vora G. J., Offenberg H. H., Palmer J. D., Zolan M. E. Isolation and characterization of rad51 orthologs from Coprinus cinereus and Lycopersicon esculentum, and phylogenetic analysis of eukaryotic recA homologs. Curr Genet. 1997 Feb;31(2):144–157. doi: 10.1007/s002940050189. [DOI] [PubMed] [Google Scholar]
  41. Stürzbecher H. W., Donzelmann B., Henning W., Knippschild U., Buchhop S. p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction. EMBO J. 1996 Apr 15;15(8):1992–2002. [PMC free article] [PubMed] [Google Scholar]
  42. Sung P. Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science. 1994 Aug 26;265(5176):1241–1243. doi: 10.1126/science.8066464. [DOI] [PubMed] [Google Scholar]
  43. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. Woods W. G., Dyall-Smith M. L. Construction and analysis of a recombination-deficient (radA) mutant of Haloferax volcanii. Mol Microbiol. 1997 Feb;23(4):791–797. doi: 10.1046/j.1365-2958.1997.2651626.x. [DOI] [PubMed] [Google Scholar]
  47. Xia S. J., Shammas M. A., Shmookler Reis R. J. Elevated recombination in immortal human cells is mediated by HsRAD51 recombinase. Mol Cell Biol. 1997 Dec;17(12):7151–7158. doi: 10.1128/mcb.17.12.7151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yasuda L. F., Yao M. C. Short inverted repeats at a free end signal large palindromic DNA formation in Tetrahymena. Cell. 1991 Nov 1;67(3):505–516. doi: 10.1016/0092-8674(91)90525-4. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES