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. 1994 Aug 2;91(16):7618–7622. doi: 10.1073/pnas.91.16.7618

The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA.

A Stasiak 1, I R Tsaneva 1, S C West 1, C J Benson 1, X Yu 1, E H Egelman 1
PMCID: PMC44453  PMID: 8052630

Abstract

The RuvB protein is induced in Escherichia coli as part of the SOS response to DNA damage. It is required for genetic recombination and the postreplication repair of DNA. In vitro, the RuvB protein promotes the branch migration of Holliday junctions and has a DNA helicase activity in reactions that require ATP hydrolysis. We have used electron microscopy, image analysis, and three-dimensional reconstruction to show that the RuvB protein, in the presence of ATP, forms a dodecamer on double-stranded DNA in which two stacked hexameric rings encircle the DNA and are oriented in opposite directions with D6 symmetry. Although helicases are ubiquitous and essential for many aspects of DNA repair, replication, and transcription, three-dimensional reconstruction of a helicase has not yet been reported, to our knowledge. The structural arrangement that is seen may be common to other helicases, such as the simian virus 40 large tumor antigen.

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Selected References

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

  1. Bennett R. J., Dunderdale H. J., West S. C. Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion. Cell. 1993 Sep 24;74(6):1021–1031. doi: 10.1016/0092-8674(93)90724-5. [DOI] [PubMed] [Google Scholar]
  2. Benson F. E., Illing G. T., Sharples G. J., Lloyd R. G. Nucleotide sequencing of the ruv region of Escherichia coli K-12 reveals a LexA regulated operon encoding two genes. Nucleic Acids Res. 1988 Feb 25;16(4):1541–1549. doi: 10.1093/nar/16.4.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boehmer P. E., Dodson M. S., Lehman I. R. The herpes simplex virus type-1 origin binding protein. DNA helicase activity. J Biol Chem. 1993 Jan 15;268(2):1220–1225. [PubMed] [Google Scholar]
  4. Dodson M. S., Lehman I. R. Association of DNA helicase and primase activities with a subassembly of the herpes simplex virus 1 helicase-primase composed of the UL5 and UL52 gene products. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1105–1109. doi: 10.1073/pnas.88.4.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dunderdale H. J., Benson F. E., Parsons C. A., Sharples G. J., Lloyd R. G., West S. C. Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins. Nature. 1991 Dec 19;354(6354):506–510. doi: 10.1038/354506a0. [DOI] [PubMed] [Google Scholar]
  6. Frank J., Bretaudiere J. P., Carazo J. M., Verschoor A., Wagenknecht T. Classification of images of biomolecular assemblies: a study of ribosomes and ribosomal subunits of Escherichia coli. J Microsc. 1988 May;150(Pt 2):99–115. doi: 10.1111/j.1365-2818.1988.tb04602.x. [DOI] [PubMed] [Google Scholar]
  7. Geiselmann J., Seifried S. E., Yager T. D., Liang C., von Hippel P. H. Physical properties of the Escherichia coli transcription termination factor rho. 2. Quaternary structure of the rho hexamer. Biochemistry. 1992 Jan 14;31(1):121–132. doi: 10.1021/bi00116a018. [DOI] [PubMed] [Google Scholar]
  8. Geiselmann J., Yager T. D., Gill S. C., Calmettes P., von Hippel P. H. Physical properties of the Escherichia coli transcription termination factor rho. 1. Association states and geometry of the rho hexamer. Biochemistry. 1992 Jan 14;31(1):111–121. doi: 10.1021/bi00116a017. [DOI] [PubMed] [Google Scholar]
  9. Geiselmann J., Yager T. D., von Hippel P. H. Functional interactions of ligand cofactors with Escherichia coli transcription termination factor rho. II. Binding of RNA. Protein Sci. 1992 Jul;1(7):861–873. doi: 10.1002/pro.5560010704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gogol E. P., Seifried S. E., von Hippel P. H. Structure and assembly of the Escherichia coli transcription termination factor rho and its interaction with RNA. I. Cryoelectron microscopic studies. J Mol Biol. 1991 Oct 20;221(4):1127–1138. doi: 10.1016/0022-2836(91)90923-t. [DOI] [PubMed] [Google Scholar]
  11. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Iwasaki H., Takahagi M., Shiba T., Nakata A., Shinagawa H. Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure. EMBO J. 1991 Dec;10(13):4381–4389. doi: 10.1002/j.1460-2075.1991.tb05016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lahue E. E., Matson S. W. Escherichia coli DNA helicase I catalyzes a unidirectional and highly processive unwinding reaction. J Biol Chem. 1988 Mar 5;263(7):3208–3215. [PubMed] [Google Scholar]
  14. Lloyd R. G., Sharples G. J. Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli. Nucleic Acids Res. 1993 Apr 25;21(8):1719–1725. doi: 10.1093/nar/21.8.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lohman T. M. Escherichia coli DNA helicases: mechanisms of DNA unwinding. Mol Microbiol. 1992 Jan;6(1):5–14. doi: 10.1111/j.1365-2958.1992.tb00831.x. [DOI] [PubMed] [Google Scholar]
  16. Mastrangelo I. A., Hough P. V., Wall J. S., Dodson M., Dean F. B., Hurwitz J. ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication. Nature. 1989 Apr 20;338(6217):658–662. doi: 10.1038/338658a0. [DOI] [PubMed] [Google Scholar]
  17. Müller B., Tsaneva I. R., West S. C. Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins. I. Comparison of RuvAB- and RuvB-mediated reactions. J Biol Chem. 1993 Aug 15;268(23):17179–17184. [PubMed] [Google Scholar]
  18. Müller B., Tsaneva I. R., West S. C. Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins. II. Interaction of RuvB with DNA. J Biol Chem. 1993 Aug 15;268(23):17185–17189. [PubMed] [Google Scholar]
  19. Oh E. Y., Grossman L. Characterization of the helicase activity of the Escherichia coli UvrAB protein complex. J Biol Chem. 1989 Jan 15;264(2):1336–1343. [PubMed] [Google Scholar]
  20. Parsons C. A., Tsaneva I., Lloyd R. G., West S. C. Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5452–5456. doi: 10.1073/pnas.89.12.5452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Parsons C. A., West S. C. Formation of a RuvAB-Holliday junction complex in vitro. J Mol Biol. 1993 Jul 20;232(2):397–405. doi: 10.1006/jmbi.1993.1399. [DOI] [PubMed] [Google Scholar]
  22. Penczek P., Radermacher M., Frank J. Three-dimensional reconstruction of single particles embedded in ice. Ultramicroscopy. 1992 Jan;40(1):33–53. [PubMed] [Google Scholar]
  23. Reha-Krantz L. J., Hurwitz J. The dnaB gene product of Escherichia coli. I. Purification, homogeneity, and physical properties. J Biol Chem. 1978 Jun 10;253(11):4043–4050. [PubMed] [Google Scholar]
  24. Rozen F., Edery I., Meerovitch K., Dever T. E., Merrick W. C., Sonenberg N. Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol. 1990 Mar;10(3):1134–1144. doi: 10.1128/mcb.10.3.1134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sharples G. J., Benson F. E., Illing G. T., Lloyd R. G. Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination. Mol Gen Genet. 1990 Apr;221(2):219–226. doi: 10.1007/BF00261724. [DOI] [PubMed] [Google Scholar]
  26. Shiba T., Iwasaki H., Nakata A., Shinagawa H. SOS-inducible DNA repair proteins, RuvA and RuvB, of Escherichia coli: functional interactions between RuvA and RuvB for ATP hydrolysis and renaturation of the cruciform structure in supercoiled DNA. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8445–8449. doi: 10.1073/pnas.88.19.8445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shinagawa H., Makino K., Amemura M., Kimura S., Iwasaki H., Nakata A. Structure and regulation of the Escherichia coli ruv operon involved in DNA repair and recombination. J Bacteriol. 1988 Sep;170(9):4322–4329. doi: 10.1128/jb.170.9.4322-4329.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tsaneva I. R., Illing G., Lloyd R. G., West S. C. Purification and properties of the RuvA and RuvB proteins of Escherichia coli. Mol Gen Genet. 1992 Oct;235(1):1–10. doi: 10.1007/BF00286175. [DOI] [PubMed] [Google Scholar]
  29. Tsaneva I. R., Müller B., West S. C. ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E. coli. Cell. 1992 Jun 26;69(7):1171–1180. doi: 10.1016/0092-8674(92)90638-s. [DOI] [PubMed] [Google Scholar]
  30. Tsaneva I. R., Müller B., West S. C. RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1315–1319. doi: 10.1073/pnas.90.4.1315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wall J. S., Hainfeld J. F. Mass mapping with the scanning transmission electron microscope. Annu Rev Biophys Biophys Chem. 1986;15:355–376. doi: 10.1146/annurev.bb.15.060186.002035. [DOI] [PubMed] [Google Scholar]
  32. West S. C. The processing of recombination intermediates: mechanistic insights from studies of bacterial proteins. Cell. 1994 Jan 14;76(1):9–15. doi: 10.1016/0092-8674(94)90168-6. [DOI] [PubMed] [Google Scholar]

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