1887

Abstract

RecG is a helicase that is conserved in nearly all bacterial species. The prototypical RecG promotes regression of stalled replication forks, participates in DNA recombination and DNA repair, and prevents aberrant replication. RecG (RecG) is a DNA-dependent ATPase that unwinds a variety of DNA substrates, although its preferred substrate is a Holliday junction. Here, we performed site-directed mutagenesis of selected residues in the wedge domain and motifs Q, I, Ib and VI of RecG. Three of the 10 substitution mutations engineered were detected previously as naturally occurring SNPs in the gene encoding RecG. Alanine substitution mutations at residues Q292, F286, K321 and R627 abolished the RecG unwinding activity, whilst RecG F99A, P285S and T408A mutants exhibited ~25–50 % lower unwinding activity than WT. We also found that RecG bound ATP in the absence of a DNA cofactor.

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2014-01-01
2020-01-18
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References

  1. Arnold K., Bordoli L., Kopp J., Schwede T..( 2006;). The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics22:195–201 [CrossRef][PubMed]
    [Google Scholar]
  2. Asai T., Kogoma T..( 1994;). Roles of ruvA, ruvC and recG gene functions in normal and DNA damage-inducible replication of the Escherichia coli chromosome. Genetics137:895–902[PubMed]
    [Google Scholar]
  3. Bifani P. J., Mathema B., Kurepina N. E., Kreiswirth B. N..( 2002;). Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol10:45–52 [CrossRef][PubMed]
    [Google Scholar]
  4. Briggs G. S., Mahdi A. A., Wen Q., Lloyd R. G..( 2005;). DNA binding by the substrate specificity (wedge) domain of RecG helicase suggests a role in processivity. J Biol Chem280:13921–13927 [CrossRef][PubMed]
    [Google Scholar]
  5. Brosh R. M. Jr, Opresko P. L., Bohr V. A..( 2006;). Enzymatic mechanism of the WRN helicase/nuclease. Methods Enzymol409:52–85 [CrossRef][PubMed]
    [Google Scholar]
  6. Cordin O., Tanner N. K., Doère M., Linder P., Banroques J..( 2004;). The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity. EMBO J23:2478–2487 [CrossRef][PubMed]
    [Google Scholar]
  7. Deyrup A. T., Krishnan S., Cockburn B. N., Schwartz N. B..( 1998;). Deletion and site-directed mutagenesis of the ATP-binding motif (P-loop) in the bifunctional murine ATP-sulfurylase/adenosine 5′-phosphosulfate kinase enzyme. J Biol Chem273:9450–9456 [CrossRef][PubMed]
    [Google Scholar]
  8. Dos Vultos T., Mestre O., Rauzier J., Golec M., Rastogi N., Rasolofo V., Tonjum T., Sola C., Matic I., Gicquel B..( 2008;). Evolution and diversity of clonal bacteria: the paradigm of Mycobacterium tuberculosis.. PLoS ONE3:e1538 [CrossRef][PubMed]
    [Google Scholar]
  9. Dos Vultos T., Mestre O., Tønjum T., Gicquel B..( 2009;). DNA repair in Mycobacterium tuberculosis revisited. FEMS Microbiol Rev33:471–487 [CrossRef][PubMed]
    [Google Scholar]
  10. Ebrahimi-Rad M., Bifani P., Martin C., Kremer K., Samper S., Rauzier J., Kreiswirth B., Blazquez J., Jouan M..& other authors ( 2003;). Mutations in putative mutator genes of Mycobacterium tuberculosis strains of the W-Beijing family. Emerg Infect Dis9:838–845 [CrossRef][PubMed]
    [Google Scholar]
  11. Edgar R. C..( 2004;). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res32:1792–1797 [CrossRef][PubMed]
    [Google Scholar]
  12. Ehrt S., Schnappinger D..( 2009;). Mycobacterial survival strategies in the phagosome: defence against host stresses. Cell Microbiol11:1170–1178 [CrossRef][PubMed]
    [Google Scholar]
  13. Elles L. M., Uhlenbeck O. C..( 2008;). Mutation of the arginine finger in the active site of Escherichia coli DbpA abolishes ATPase and helicase activity and confers a dominant slow growth phenotype. Nucleic Acids Res36:41–50 [CrossRef][PubMed]
    [Google Scholar]
  14. European Concerted Action on New Generation Genetic Markers and Techniques for the Epidemiology and Control of Tuberculosis( 2006;). Beijing/W genotype Mycobacterium tuberculosis and drug resistance. Emerg Infect Dis12:736–743 [CrossRef][PubMed]
    [Google Scholar]
  15. Filliol I., Motiwala A. S., Cavatore M., Qi W., Hazbón M. H., Bobadilla del Valle M., Fyfe J., García-García L., Rastogi N..& other authors ( 2006;). Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set. J Bacteriol188:759–772 [CrossRef][PubMed]
    [Google Scholar]
  16. Gagneux S..( 2009;). Strain variation and evolution. Mycobacteria: Genomics and Molecular Biology1–18 Parish T., Brown A.. Norwich: Caister Academic Press;
    [Google Scholar]
  17. Gamulin V., Cetkovic H., Ahel I..( 2004;). Identification of a promoter motif regulating the major DNA damage response mechanism of Mycobacterium tuberculosis. FEMS Microbiol Lett238:57–63[PubMed]
    [Google Scholar]
  18. Gorbalenya A. E., Koonin E. V..( 1993;). Helicases: amino acid sequence comparisons and structure-function relationships. Curr Opin Struct Biol3:419–429 [CrossRef]
    [Google Scholar]
  19. Gorna A. E., Bowater R. P., Dziadek J..( 2010;). DNA repair systems and the pathogenesis of Mycobacterium tuberculosis: varying activities at different stages of infection. Clin Sci (Lond)119:187–202 [CrossRef][PubMed]
    [Google Scholar]
  20. Hall M. C., Matson S. W..( 1999;). Helicase motifs: the engine that powers DNA unwinding. Mol Microbiol34:867–877 [CrossRef][PubMed]
    [Google Scholar]
  21. Kornberg A., Scott J. F., Bertsch L. L..( 1978;). ATP utilization by rep protein in the catalytic separation of DNA strands at a replicating fork. J Biol Chem253:3298–3304[PubMed]
    [Google Scholar]
  22. Lloyd R. G., Sharples G. J..( 1993;). Dissociation of synthetic Holliday junctions by E. coli RecG protein. EMBO J12:17–22[PubMed]
    [Google Scholar]
  23. Lohman T. M., Tomko E. J., Wu C. G..( 2008;). Non-hexameric DNA helicases and translocases: mechanisms and regulation. Nat Rev Mol Cell Biol9:391–401 [CrossRef][PubMed]
    [Google Scholar]
  24. Mahdi A. A., Briggs G. S., Sharples G. J., Wen Q., Lloyd R. G..( 2003;). A model for dsDNA translocation revealed by a structural motif common to RecG and Mfd proteins. EMBO J22:724–734 [CrossRef][PubMed]
    [Google Scholar]
  25. Mahdi A. A., Briggs G. S., Lloyd R. G..( 2012;). Modulation of DNA damage tolerance in Escherichia coli recG and ruv strains by mutations affecting PriB, the ribosome and RNA polymerase. Mol Microbiol86:675–691 [CrossRef][PubMed]
    [Google Scholar]
  26. McGlynn P., Lloyd R. G..( 2002;). Genome stability and the processing of damaged replication forks by RecG. Trends Genet18:413–419 [CrossRef][PubMed]
    [Google Scholar]
  27. McGlynn P., Mahdi A. A., Lloyd R. G..( 2000;). Characterisation of the catalytically active form of RecG helicase. Nucleic Acids Res28:2324–2332 [CrossRef][PubMed]
    [Google Scholar]
  28. Mestre O., Luo T., Dos Vultos T., Kremer K., Murray A., Namouchi A., Jackson C., Rauzier J., Bifani P..& other authors ( 2011;). Phylogeny of Mycobacterium tuberculosis Beijing strains constructed from polymorphisms in genes involved in DNA replication, recombination and repair. PLoS ONE6:e16020 [CrossRef][PubMed]
    [Google Scholar]
  29. Myong S., Bruno M. M., Pyle A. M., Ha T..( 2007;). Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase. Science317:513–516 [CrossRef][PubMed]
    [Google Scholar]
  30. O’Reilly E. K., Kreuzer K. N..( 2004;). Isolation of SOS constitutive mutants of Escherichia coli. J Bacteriol186:7149–7160 [CrossRef][PubMed]
    [Google Scholar]
  31. Pause A., Sonenberg N..( 1992;). Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J11:2643–2654[PubMed]
    [Google Scholar]
  32. Pyle A. M..( 2008;). Translocation and unwinding mechanisms of RNA and DNA helicases. Annu Rev Biophys37:317–336 [CrossRef][PubMed]
    [Google Scholar]
  33. Rachman H., Strong M., Ulrichs T., Grode L., Schuchhardt J., Mollenkopf H., Kosmiadi G. A., Eisenberg D., Kaufmann S. H..( 2006;). Unique transcriptome signature of Mycobacterium tuberculosis in pulmonary tuberculosis. Infect Immun74:1233–1242 [CrossRef][PubMed]
    [Google Scholar]
  34. Ramaswamy S. V., Reich R., Dou S. J., Jasperse L., Pan X., Wanger A., Quitugua T., Graviss E. A..( 2003;). Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother47:1241–1250 [CrossRef][PubMed]
    [Google Scholar]
  35. Rudolph C. J., Upton A. L., Harris L., Lloyd R. G..( 2009a;). Pathological replication in cells lacking RecG DNA translocase. Mol Microbiol73:352–366 [CrossRef][PubMed]
    [Google Scholar]
  36. Rudolph C. J., Upton A. L., Lloyd R. G..( 2009b;). Replication fork collisions cause pathological chromosomal amplification in cells lacking RecG DNA translocase. Mol Microbiol74:940–955 [CrossRef][PubMed]
    [Google Scholar]
  37. Rudolph C. J., Upton A. L., Briggs G. S., Lloyd R. G..( 2010;). Is RecG a general guardian of the bacterial genome. DNA Repair (Amst)9:210–223 [CrossRef][PubMed]
    [Google Scholar]
  38. Sayers E. W., Barrett T., Benson D. A., Bolton E., Bryant S. H., Canese K., Chetvernin V., Church D. M., Dicuccio M..& other authors ( 2012;). Database resources of the National Center for Biotechnology Information. Nucleic Acids Res40:Database issueD13–D25 [CrossRef][PubMed]
    [Google Scholar]
  39. Schnappinger D., Ehrt S., Voskuil M. I., Liu Y., Mangan J. A., Monahan I. M., Dolganov G., Efron B., Butcher P. D..& other authors ( 2003;). Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J Exp Med198:693–704 [CrossRef][PubMed]
    [Google Scholar]
  40. Sharples G. J., Whitby M. C., Ryder L., Lloyd R. G..( 1994;). A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions. Nucleic Acids Res22:308–313 [CrossRef][PubMed]
    [Google Scholar]
  41. Sharples G. J., Ingleston S. M., Lloyd R. G..( 1999;). Holliday junction processing in bacteria: insights from the evolutionary conservation of RuvABC, RecG, and RusA. J Bacteriol181:5543–5550[PubMed]
    [Google Scholar]
  42. Singleton M. R., Scaife S., Wigley D. B..( 2001;). Structural analysis of DNA replication fork reversal by RecG. Cell107:79–89 [CrossRef][PubMed]
    [Google Scholar]
  43. Smollett K. L., Smith K. M., Kahramanoglou C., Arnvig K. B., Buxton R. S., Davis E. O..( 2012;). Global analysis of the regulon of the transcriptional repressor LexA, a key component of SOS response in Mycobacterium tuberculosis. J Biol Chem287:22004–22014 [CrossRef][PubMed]
    [Google Scholar]
  44. Stallings C. L., Glickman M. S..( 2010;). Is Mycobacterium tuberculosis stressed out? A critical assessment of the genetic evidence. Microbes Infect12:1091–1101 [CrossRef][PubMed]
    [Google Scholar]
  45. Tanner N. K., Cordin O., Banroques J., Doère M., Linder P..( 2003;). The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis. Mol Cell11:127–138 [CrossRef][PubMed]
    [Google Scholar]
  46. Telenti A., Imboden P., Marchesi F., Matter L., Schopfer K., Bodmer T., Lowrie D., Colston M. J., Cole S..( 1993;). Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet341:647–651 [CrossRef][PubMed]
    [Google Scholar]
  47. Thakur R. S., Basavaraju S., Somyajit K., Jain A., Subramanya S., Muniyappa K., Nagaraju G..( 2013;). Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination. FEBS J280:1841–1860 [CrossRef][PubMed]
    [Google Scholar]
  48. Tuteja N., Tuteja R..( 2004;). Unraveling DNA helicases. Motif, structure, mechanism and function. Eur J Biochem271:1849–1863 [CrossRef][PubMed]
    [Google Scholar]
  49. Velankar S. S., Soultanas P., Dillingham M. S., Subramanya H. S., Wigley D. B..( 1999;). Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell97:75–84 [CrossRef][PubMed]
    [Google Scholar]
  50. Voskuil M. I., Bartek I. L., Visconti K., Schoolnik G. K..( 2011;). The response of Mycobacterium tuberculosis to reactive oxygen and nitrogen species. Front Microbiol2:105 [CrossRef][PubMed]
    [Google Scholar]
  51. Warner D. F., Tønjum T., Mizrahi V..( 2013;). DNA metabolism in mycobacterial pathogenesis. Curr Top Microbiol Immunol [CrossRef][PubMed]
    [Google Scholar]
  52. Waterhouse A. M., Procter J. B., Martin D. M., Clamp M., Barton G. J..( 2009;). Jalview Version 2 – a multiple sequence alignment editor and analysis workbench. Bioinformatics25:1189–1191 [CrossRef][PubMed]
    [Google Scholar]
  53. Whitby M. C., Lloyd R. G..( 1998;). Targeting Holliday junctions by the RecG branch migration protein of Escherichia coli. J Biol Chem273:19729–19739 [CrossRef][PubMed]
    [Google Scholar]
  54. WHO( 2011;). Global Tuberculosis Control 2011 Geneva: World Health Organization;
    [Google Scholar]
  55. WHO( 2012;). Global Tuberculosis Report 2012 Geneva: World Health Organization;
    [Google Scholar]
  56. Wu Y., Sommers J. A., Loiland J. A., Kitao H., Kuper J., Kisker C., Brosh R. M. Jr.( 2012;). The Q motif of Fanconi anemia group J protein (FANCJ) DNA helicase regulates its dimerization, DNA binding, and DNA repair function. J Biol Chem287:21699–21716 [CrossRef][PubMed]
    [Google Scholar]
  57. Zaczek A., Brzostek A., Augustynowicz-Kopec E., Zwolska Z., Dziadek J..( 2009;). Genetic evaluation of relationship between mutations in rpoB and resistance of Mycobacterium tuberculosis to rifampin. BMC Microbiol9:10 [CrossRef][PubMed]
    [Google Scholar]
  58. Zegeye E. D., Balasingham S. V., Laerdahl J. K., Homberset H., Tønjum T..( 2012;). Mycobacterium tuberculosis RecG binds and unwinds model DNA substrates with a preference for Holliday junctions. Microbiology158:1982–1993 [CrossRef][PubMed]
    [Google Scholar]
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