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. 1997 Sep 1;16(17):5198–5206. doi: 10.1093/emboj/16.17.5198

The human Rad51 protein: polarity of strand transfer and stimulation by hRP-A.

P Baumann 1, S C West 1
PMCID: PMC1170152  PMID: 9311980

Abstract

The human Rad51 protein is homologous to the RecA protein and catalyses homologous pairing and strand transfer reactions in vitro. Using single-stranded circular and homologous linear duplex DNA, we show that hRad51 forms stable joint molecules by transfer of the 5' end of the complementary strand of the linear duplex to the ssDNA. The polarity of strand transfer is therefore 3' to 5', defined relative to the ssDNA on which hRad51 initiates filament formation. This polarity is opposite to that observed with RecA. Homologous pairing and strand transfer require stoichiometric amounts of hRad51, corresponding to one hRad51 monomer per three nucleotides of ssDNA. Joint molecules are not observed when the protein is present in limiting or excessive amounts. The human ssDNA binding-protein, hRP-A, stimulates hRad51-mediated reactions. Its effect is consistent with a role in the removal of secondary structures from ssDNA, thereby facilitating the formation of continuous Rad51 filaments.

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Selected References

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  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. Ajimura M., Leem S. H., Ogawa H. Identification of new genes required for meiotic recombination in Saccharomyces cerevisiae. Genetics. 1993 Jan;133(1):51–66. doi: 10.1093/genetics/133.1.51. [DOI] [PMC free article] [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. Barlow A. L., Benson F. E., West S. C., Hultén M. A. Distribution of the Rad51 recombinase in human and mouse spermatocytes. EMBO J. 1997 Sep 1;16(17):5207–5215. doi: 10.1093/emboj/16.17.5207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  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. 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]
  10. Chow S. A., Rao B. J., Radding C. M. Reversibility of strand invasion promoted by recA protein and its inhibition by Escherichia coli single-stranded DNA-binding protein or phage T4 gene 32 protein. J Biol Chem. 1988 Jan 5;263(1):200–209. [PubMed] [Google Scholar]
  11. Cox M. M., Lehman I. R. Directionality and polarity in recA protein-promoted branch migration. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6018–6022. doi: 10.1073/pnas.78.10.6018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cox M. M., Soltis D. A., Livneh Z., Lehman I. R. On the role of single-stranded DNA binding protein in recA protein-promoted DNA strand exchange. J Biol Chem. 1983 Feb 25;258(4):2577–2585. [PubMed] [Google Scholar]
  13. Gupta R. C., Bazemore L. R., Golub E. I., Radding C. M. Activities of human recombination protein Rad51. Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):463–468. doi: 10.1073/pnas.94.2.463. [DOI] [PMC free article] [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. Hays S. L., Firmenich A. A., Berg P. Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6925–6929. doi: 10.1073/pnas.92.15.6925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henricksen L. A., Umbricht C. B., Wold M. S. Recombinant replication protein A: expression, complex formation, and functional characterization. J Biol Chem. 1994 Apr 15;269(15):11121–11132. [PubMed] [Google Scholar]
  17. Heuser J., Griffith J. Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy. J Mol Biol. 1989 Dec 5;210(3):473–484. doi: 10.1016/0022-2836(89)90124-1. [DOI] [PubMed] [Google Scholar]
  18. Howard-Flanders P., West S. C., Stasiak A. Role of RecA protein spiral filaments in genetic recombination. Nature. 1984 May 17;309(5965):215–219. doi: 10.1038/309215a0. [DOI] [PubMed] [Google Scholar]
  19. Ivanov E. L., Korolev V. G., Fabre F. XRS2, a DNA repair gene of Saccharomyces cerevisiae, is needed for meiotic recombination. Genetics. 1992 Nov;132(3):651–664. doi: 10.1093/genetics/132.3.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jiang H., Xie Y., Houston P., Stemke-Hale K., Mortensen U. H., Rothstein R., Kodadek T. Direct association between the yeast Rad51 and Rad54 recombination proteins. J Biol Chem. 1996 Dec 27;271(52):33181–33186. doi: 10.1074/jbc.271.52.33181. [DOI] [PubMed] [Google Scholar]
  21. Kovalenko O. V., Plug A. W., Haaf T., Gonda D. K., Ashley T., Ward D. C., Radding C. M., Golub E. I. Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2958–2963. doi: 10.1073/pnas.93.7.2958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kowalczykowski S. C., Krupp R. A. Effects of Escherichia coli SSB protein on the single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein. Evidence that SSB protein facilitates the binding of RecA protein to regions of secondary structure within single-stranded DNA. J Mol Biol. 1987 Jan 5;193(1):97–113. doi: 10.1016/0022-2836(87)90630-9. [DOI] [PubMed] [Google Scholar]
  23. 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]
  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. McEntee K., Weinstock G. M., Lehman I. R. Binding of the recA protein of Escherichia coli to single- and double-stranded DNA. J Biol Chem. 1981 Aug 25;256(16):8835–8844. [PubMed] [Google Scholar]
  26. Menetski J. P., Kowalczykowski S. C. Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors. J Mol Biol. 1985 Jan 20;181(2):281–295. doi: 10.1016/0022-2836(85)90092-0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Plug A. W., Xu J., Reddy G., Golub E. I., Ashley T. Presynaptic association of Rad51 protein with selected sites in meiotic chromatin. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5920–5924. doi: 10.1073/pnas.93.12.5920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roca A. I., Cox M. M. RecA protein: structure, function, and role in recombinational DNA repair. Prog Nucleic Acid Res Mol Biol. 1997;56:129–223. doi: 10.1016/s0079-6603(08)61005-3. [DOI] [PubMed] [Google Scholar]
  30. Scully R., Chen J., Plug A., Xiao Y., Weaver D., Feunteun J., Ashley T., Livingston D. M. Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell. 1997 Jan 24;88(2):265–275. doi: 10.1016/s0092-8674(00)81847-4. [DOI] [PubMed] [Google Scholar]
  31. Shah R., Bennett R. J., West S. C. Genetic recombination in E. coli: RuvC protein cleaves Holliday junctions at resolution hotspots in vitro. Cell. 1994 Dec 2;79(5):853–864. doi: 10.1016/0092-8674(94)90074-4. [DOI] [PubMed] [Google Scholar]
  32. Shen Z., Cloud K. G., Chen D. J., Park M. S. Specific interactions between the human RAD51 and RAD52 proteins. J Biol Chem. 1996 Jan 5;271(1):148–152. doi: 10.1074/jbc.271.1.148. [DOI] [PubMed] [Google Scholar]
  33. Shen Z., Pardington-Purtymun P. E., Comeaux J. C., Moyzis R. K., Chen D. J. UBL1, a human ubiquitin-like protein associating with human RAD51/RAD52 proteins. Genomics. 1996 Sep 1;36(2):271–279. doi: 10.1006/geno.1996.0462. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. Terasawa M., Shinohara A., Hotta Y., Ogawa H., Ogawa T. Localization of RecA-like recombination proteins on chromosomes of the lily at various meiotic stages. Genes Dev. 1995 Apr 15;9(8):925–934. doi: 10.1101/gad.9.8.925. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. West S. C., Cassuto E., Howard-Flanders P. Heteroduplex formation by recA protein: polarity of strand exchanges. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6149–6153. doi: 10.1073/pnas.78.10.6149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. West S. C., Cassuto E., Howard-Flanders P. Postreplication repair in E. coli: strand exchange reactions of gapped DNA by RecA protein. Mol Gen Genet. 1982;187(2):209–217. doi: 10.1007/BF00331119. [DOI] [PubMed] [Google Scholar]
  42. Yoshimura Y., Morita T., Yamamoto A., Matsushiro A. Cloning and sequence of the human RecA-like gene cDNA. Nucleic Acids Res. 1993 Apr 11;21(7):1665–1665. doi: 10.1093/nar/21.7.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yu X., Egelman E. H. Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible. J Mol Biol. 1992 Sep 5;227(1):334–346. doi: 10.1016/0022-2836(92)90702-l. [DOI] [PubMed] [Google Scholar]

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