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. 1994 Dec 1;13(23):5764–5771. doi: 10.1002/j.1460-2075.1994.tb06914.x

Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA.

F E Benson 1, A Stasiak 1, S C West 1
PMCID: PMC395542  PMID: 7988572

Abstract

In bacteria, genetic recombination is catalysed by RecA protein, the product of the recA gene. A human gene that shares homology with Escherichia coli recA (and its yeast homologue RAD51) has been cloned from a testis cDNA library, and its 37 kDa product (hRad51) purified to homogeneity. The human Rad51 protein binds to single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using a topological assay, we demonstrate that hRad51 underwinds duplex DNA, in a reaction dependent upon the presence of ATP or its non-hydrolysable analogue ATP gamma S. Complexes formed with single- and double-stranded DNA have been observed by electron microscopy following negative staining. With nicked duplex DNA, hRad51 forms helical nucleoprotein filaments which exhibit the striated appearance characteristic of RecA or yeast Rad51 filaments. Contour length measurements indicate that the DNA is underwound and extended within the nucleoprotein complex. In contrast to yeast Rad51 protein, human Rad51 forms filaments with single-stranded DNA in the presence of ATP/ATP gamma S. These resemble the inactive form of the RecA filament which is observed in the absence of a nucleotide cofactor.

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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. Adams D. E., Tsaneva I. R., West S. C. Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9901–9905. doi: 10.1073/pnas.91.21.9901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benedict R. C., Kowalczykowski S. C. Increase of the DNA strand assimilation activity of recA protein by removal of the C terminus and structure-function studies of the resulting protein fragment. J Biol Chem. 1988 Oct 25;263(30):15513–15520. [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. Bishop D. K., Park D., Xu L., Kleckner N. DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell. 1992 May 1;69(3):439–456. doi: 10.1016/0092-8674(92)90446-j. [DOI] [PubMed] [Google Scholar]
  6. CLARK A. J., MARGULIES A. D. ISOLATION AND CHARACTERIZATION OF RECOMBINATION-DEFICIENT MUTANTS OF ESCHERICHIA COLI K12. Proc Natl Acad Sci U S A. 1965 Feb;53:451–459. doi: 10.1073/pnas.53.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cheng R., Baker T. I., Cords C. E., Radloff R. J. mei-3, a recombination and repair gene of Neurospora crassa, encodes a RecA-like protein. Mutat Res. 1993 Oct;294(3):223–234. doi: 10.1016/0921-8777(93)90005-2. [DOI] [PubMed] [Google Scholar]
  8. Cox M. M., Lehman I. R. Enzymes of general recombination. Annu Rev Biochem. 1987;56:229–262. doi: 10.1146/annurev.bi.56.070187.001305. [DOI] [PubMed] [Google Scholar]
  9. Cox M. M., McEntee K., Lehman I. R. A simple and rapid procedure for the large scale purification of the recA protein of Escherichia coli. J Biol Chem. 1981 May 10;256(9):4676–4678. [PubMed] [Google Scholar]
  10. Di Capua E., Engel A., Stasiak A., Koller T. Characterization of complexes between recA protein and duplex DNA by electron microscopy. J Mol Biol. 1982 May 5;157(1):87–103. doi: 10.1016/0022-2836(82)90514-9. [DOI] [PubMed] [Google Scholar]
  11. Flory J., Radding C. M. Visualization of recA protein and its association with DNA: a priming effect of single-strand-binding protein. Cell. 1982 Apr;28(4):747–756. doi: 10.1016/0092-8674(82)90054-x. [DOI] [PubMed] [Google Scholar]
  12. Friedman K. D., Rosen N. L., Newman P. J., Montgomery R. R. Enzymatic amplification of specific cDNA inserts from lambda gt11 libraries. Nucleic Acids Res. 1988 Sep 12;16(17):8718–8718. doi: 10.1093/nar/16.17.8718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harris L. D., Griffith J. Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein. J Biol Chem. 1987 Jul 5;262(19):9285–9292. [PubMed] [Google Scholar]
  14. 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]
  15. Iwabuchi M., Shibata T., Ohtani T., Natori M., Ando T. ATP-dependent unwinding of the double helix and extensive supercoiling by Escherichia coli recA protein in the presence of topoisomerase. J Biol Chem. 1983 Oct 25;258(20):12394–12404. [PubMed] [Google Scholar]
  16. Kodadek T., Wong M. L., Alberts B. M. The mechanism of homologous DNA strand exchange catalyzed by the bacteriophage T4 uvsX and gene 32 proteins. J Biol Chem. 1988 Jul 5;263(19):9427–9436. [PubMed] [Google Scholar]
  17. Kowalczykowski S. C. Biochemistry of genetic recombination: energetics and mechanism of DNA strand exchange. Annu Rev Biophys Biophys Chem. 1991;20:539–575. doi: 10.1146/annurev.bb.20.060191.002543. [DOI] [PubMed] [Google Scholar]
  18. Kowalczykowski S. C., Eggleston A. K. Homologous pairing and DNA strand-exchange proteins. Annu Rev Biochem. 1994;63:991–1043. doi: 10.1146/annurev.bi.63.070194.005015. [DOI] [PubMed] [Google Scholar]
  19. Lloyd R. G., Buckman C., Benson F. E. Genetic analysis of conjugational recombination in Escherichia coli K12 strains deficient in RecBCD enzyme. J Gen Microbiol. 1987 Sep;133(9):2531–2538. doi: 10.1099/00221287-133-9-2531. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Ogawa T., Shinohara A., Ogawa H., Tomizawa J. Functional structures of the recA protein found by chimera analysis. J Mol Biol. 1992 Aug 5;226(3):651–660. doi: 10.1016/0022-2836(92)90622-q. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Radding C. M. Helical interactions in homologous pairing and strand exchange driven by RecA protein. J Biol Chem. 1991 Mar 25;266(9):5355–5358. [PubMed] [Google Scholar]
  25. 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]
  26. Ruigrok R. W., Bohrmann B., Hewat E., Engel A., Kellenberger E., DiCapua E. The inactive form of recA protein: the 'compact' structure. EMBO J. 1993 Jan;12(1):9–16. doi: 10.1002/j.1460-2075.1993.tb05626.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Smith M. H., Cavenagh M. M., Little J. W. Mutant LexA proteins with an increased rate of in vivo cleavage. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7356–7360. doi: 10.1073/pnas.88.16.7356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stasiak A., Di Capua E., Koller T. Elongation of duplex DNA by recA protein. J Mol Biol. 1981 Sep 25;151(3):557–564. doi: 10.1016/0022-2836(81)90010-3. [DOI] [PubMed] [Google Scholar]
  32. Stasiak A., Di Capua E. The helicity of DNA in complexes with recA protein. Nature. 1982 Sep 9;299(5879):185–186. doi: 10.1038/299185a0. [DOI] [PubMed] [Google Scholar]
  33. Stasiak A., Stasiak A. Z., Koller T. Visualization of RecA-DNA complexes involved in consecutive stages of an in vitro strand exchange reaction. Cold Spring Harb Symp Quant Biol. 1984;49:561–570. doi: 10.1101/sqb.1984.049.01.063. [DOI] [PubMed] [Google Scholar]
  34. Story R. M., Steitz T. A. Structure of the recA protein-ADP complex. Nature. 1992 Jan 23;355(6358):374–376. doi: 10.1038/355374a0. [DOI] [PubMed] [Google Scholar]
  35. Story R. M., Weber I. T., Steitz T. A. The structure of the E. coli recA protein monomer and polymer. Nature. 1992 Jan 23;355(6358):318–325. doi: 10.1038/355318a0. [DOI] [PubMed] [Google Scholar]
  36. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Tateishi S., Horii T., Ogawa T., Ogawa H. C-terminal truncated Escherichia coli RecA protein RecA5327 has enhanced binding affinities to single- and double-stranded DNAs. J Mol Biol. 1992 Jan 5;223(1):115–129. doi: 10.1016/0022-2836(92)90720-5. [DOI] [PubMed] [Google Scholar]
  39. Walker G. C. Inducible DNA repair systems. Annu Rev Biochem. 1985;54:425–457. doi: 10.1146/annurev.bi.54.070185.002233. [DOI] [PubMed] [Google Scholar]
  40. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. West S. C., Cassuto E., Mursalim J., Howard-Flanders P. Recognition of duplex DNA containing single-stranded regions by recA protein. Proc Natl Acad Sci U S A. 1980 May;77(5):2569–2573. doi: 10.1073/pnas.77.5.2569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. West S. C. Enzymes and molecular mechanisms of genetic recombination. Annu Rev Biochem. 1992;61:603–640. doi: 10.1146/annurev.bi.61.070192.003131. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Yu X., Egelman E. H. DNA conformation induced by the bacteriophage T4 UvsX protein appears identical to the conformation induced by the Escherichia coli RecA protein. J Mol Biol. 1993 Jul 5;232(1):1–4. doi: 10.1006/jmbi.1993.1363. [DOI] [PubMed] [Google Scholar]
  45. Yu X., Egelman E. H. Image analysis reveals that Escherichia coli RecA protein consists of two domains. Biophys J. 1990 Mar;57(3):555–566. doi: 10.1016/S0006-3495(90)82571-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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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