Mutual interaction enables the mycobacterial plasmid pAL5000 origin binding protein RepB to recruit RepA, the plasmid replicase, to the origin Free

Abstract

The plasmid, pAL5000, is the most-studied member of a family of plasmids that are found in . Its replication is brought about by the combined action of two plasmid-encoded replication proteins, RepA and RepB. RepB has earlier been shown to be a sigma factor homologue that possesses origin-binding activity. The mechanism by which RepA functions, and its relationship with RepB, if any, has not been explored yet. In this study, we show that RepA shares a common catalytic domain, with proteins belonging to the primase-polymerase and DNA polymerase X families. We demonstrate that RepA is functionally a DNA polymerase and that mutations that alter two conserved aspartic acid residues present within the catalytic core lead to inactivation of plasmid replication. Replication of pAL5000 was shown not to depend on the host primase, and thus it is most likely that RepA is responsible for the priming act. We further demonstrate that RepA and RepB function as a pair and that the functional cooperation between the two requires physical contact. The C-terminal domain of RepA, which is structurally a helical bundle, is responsible for unwinding the origin in a site-specific manner and also for the establishment of contacts with RepB. The results presented show that RepB functions by recruiting RepA to the origin in much the same way as sigma factors recruit RNA polymerase core enzyme to promoters.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000447
2017-04-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/163/4/595.html?itemId=/content/journal/micro/10.1099/mic.0.000447&mimeType=html&fmt=ahah

References

  1. Filée J, Forterre P, Sen-Lin T, Laurent J. Evolution of DNA polymerase families: evidences for multiple gene exchange between cellular and viral proteins. J Mol Evol 2002; 54:763–773 [View Article][PubMed]
    [Google Scholar]
  2. Leipe DD, Aravind L, Koonin EV. Did DNA replication evolve twice independently?. Nucleic Acids Res 1999; 27:3389–3401 [View Article][PubMed]
    [Google Scholar]
  3. Basu A, Chawla-Sarkar M, Chakrabarti S, Das Gupta SK. Origin binding activity of the mycobacterial plasmid pAL5000 replication protein RepB is stimulated through interactions with host factors and coupled expression of repA. J Bacteriol 2002; 184:2204–2214 [View Article][PubMed]
    [Google Scholar]
  4. Stolt P, Stoker NG. Protein-DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000. J Bacteriol 1996; 178:6693–6700 [View Article][PubMed]
    [Google Scholar]
  5. Basu A, Chatterjee S, Das Gupta SK. Translational coupling to an upstream gene promotes folding of the mycobacterial plasmid pAL5000 replication protein RepB and thereby its origin binding activity. J Bacteriol 2004; 186:335–342 [View Article][PubMed]
    [Google Scholar]
  6. Stolt P, Stoker NG. Functional definition of regions necessary for replication and incompatibility in the Mycobacterium fortuitum plasmid pAL5000. Microbiology 1996; 142:2795–2802 [View Article][PubMed]
    [Google Scholar]
  7. Chak KF, James R. Characterization of the ColE9-J plasmid and analysis of its genetic organization. J Gen Microbiol 1986; 132:61–70 [View Article][PubMed]
    [Google Scholar]
  8. Basu A, Chatterjee S, Chatterjee S, Das Gupta SK. Evolutionary link between the mycobacterial plasmid pAL5000 replication protein RepB and the extracytoplasmic function family of σ factors. J Bacteriol 2012; 194:1331–1341 [View Article][PubMed]
    [Google Scholar]
  9. Han M, Yagura M, Itoh T. Specific interaction between the initiator protein (Rep) and origin of plasmid ColE2-P9. J Bacteriol 2007; 189:1061–1071 [View Article][PubMed]
    [Google Scholar]
  10. Hiraga S, Sugiyama T, Itoh T. Comparative analysis of the replicon regions of eleven ColE2-related plasmids. J Bacteriol 1994; 176:7233–7243 [View Article][PubMed]
    [Google Scholar]
  11. Owens JT, Miyake R, Murakami K, Chmura AJ, Fujita N et al. Mapping the sigma70 subunit contact sites on Escherichia coli RNA polymerase with a sigma70-conjugated chemical protease. Proc Natl Acad Sci USA 1998; 95:6021–6026 [View Article][PubMed]
    [Google Scholar]
  12. Studier FW. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 1991; 219:37–44 [View Article][PubMed]
    [Google Scholar]
  13. Snapper SB, Melton RE, Mustafa S, Kieser T, Jacobs WR Jr. Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 1990; 4:1911–1919 [View Article][PubMed]
    [Google Scholar]
  14. Chawla M, Das Gupta SK. Transposition-induced structural instability of Escherichia coli-mycobacteria shuttle vectors. Plasmid 1999; 41:135–140 [View Article][PubMed]
    [Google Scholar]
  15. Blokpoel MC, Murphy HN, O'Toole R, Wiles S, Runn ES et al. Tetracycline-inducible gene regulation in mycobacteria. Nucleic Acids Res 2005; 33:e22 [View Article][PubMed]
    [Google Scholar]
  16. Van Kessel JC, Marinelli LJ, Hatfull GF. Recombineering mycobacteria and their phages. Nat Rev Microbiol 2008; 6:851–857 [View Article][PubMed]
    [Google Scholar]
  17. Triccas JA, Parish T, Britton WJ, Gicquel B. An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis. FEMS Microbiol Lett 1998; 167:151–156 [View Article][PubMed]
    [Google Scholar]
  18. Ghosh S, Samaddar S, Kirtania P, Das Gupta SK. A DinB ortholog enables mycobacterial growth under dTTP-limiting conditions induced by the expression of a mycobacteriophage-derived ribonucleotide reductase gene. J Bacteriol 2016; 198:352–362 [View Article]
    [Google Scholar]
  19. Castanié MP, Bergès H, Oreglia J, Prère MF, Fayet O. A set of pBR322-compatible plasmids allowing the testing of chaperone-assisted folding of proteins overexpressed in Escherichia coli. Anal Biochem 1997; 254:150–152 [View Article][PubMed]
    [Google Scholar]
  20. Bhowmik P, Das Gupta SK. Biochemical characterization of a mycobacteriophage derived DnaB ortholog reveals new insight into the evolutionary origin of DnaB helicases. PLoS One 2015; 10:e0134762 [View Article][PubMed]
    [Google Scholar]
  21. Chatterjee S, Basu A, Basu A, Das Gupta SK. DNA bending in the mycobacterial plasmid pAL5000 origin-RepB complex. J Bacteriol 2007; 189:8584–8592 [View Article][PubMed]
    [Google Scholar]
  22. Kim J, Zwieb C, Wu C, Adhya S. Bending of DNA by gene-regulatory proteins: construction and use of a DNA bending vector. Gene 1989; 85:15–23 [View Article][PubMed]
    [Google Scholar]
  23. Bandyopadhyay B, das Gupta T, Roy D, Das Gupta SK. DnaK dependence of the mycobacterial stress-responsive regulator HspR is mediated through its hydrophobic C-terminal tail. J Bacteriol 2012; 194:4688–4697 [View Article][PubMed]
    [Google Scholar]
  24. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994; 22:4673–4680 [View Article][PubMed]
    [Google Scholar]
  25. Tamura K, Peterson D, Peterson N, Stecher G, Nei M et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011; 28:2731–2739 [View Article][PubMed]
    [Google Scholar]
  26. Iyer LM, Koonin EV, Leipe DD, Aravind L. Origin and evolution of the archaeo-eukaryotic primase superfamily and related palm-domain proteins: structural insights and new members. Nucleic Acids Res 2005; 33:3875–3896 [View Article][PubMed]
    [Google Scholar]
  27. Itoh T. Replication and its regulation of ColE2 and ColE3. Adv Biophys 1986; 21:105–114 [View Article][PubMed]
    [Google Scholar]
  28. Geibel S, Banchenko S, Engel M, Lanka E, Saenger W. Structure and function of primase RepB' encoded by broad-host-range plasmid RSF1010 that replicates exclusively in leading-strand mode. Proc Natl Acad Sci USA 2009; 106:7810–7815 [View Article][PubMed]
    [Google Scholar]
  29. Beck K, Vannini A, Cramer P, Lipps G. The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis. Nucleic Acids Res 2010; 38:6707–6718 [View Article][PubMed]
    [Google Scholar]
  30. Kirk BW, Kuchta RD. Arg304 of human DNA primase is a key contributor to catalysis and NTP binding: primase and the family X polymerases share significant sequence homology. Biochemistry 1999; 38:7727–7736 [View Article][PubMed]
    [Google Scholar]
  31. Yamtich J, Sweasy JB. DNA polymerase family X: function, structure, and cellular roles. Biochimica Et Biophysica Acta 1804; 2010:1136–1150
    [Google Scholar]
  32. Lipps G, Weinzierl AO, Von Scheven G, Buchen C, Cramer P. Structure of a bifunctional DNA primase-polymerase. Nat Struct Mol Biol 2004; 11:157–162 [View Article][PubMed]
    [Google Scholar]
  33. Aravind L, Mazumder R, Vasudevan S, Koonin EV. Trends in protein evolution inferred from sequence and structure analysis. Curr Opin Struct Biol 2002; 12:392–399 [View Article][PubMed]
    [Google Scholar]
  34. Gajadeera C, Willby MJ, Green KD, Shaul P, Fridman M et al. Antimycobacterial activity of DNA intercalator inhibitors of Mycobacterium tuberculosis primase DnaG. J Antibiot 2015; 68:153–157 [View Article][PubMed]
    [Google Scholar]
  35. Itou H, Yagura M, Shirakihara Y, Itoh T. Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein. J Biol Chem 2015; 290:3601–3611 [View Article][PubMed]
    [Google Scholar]
  36. Ranes MG, Rauzier J, Lagranderie M, Gheorghiu M, Gicquel B. Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a “mini” mycobacterium-Escherichia coli shuttle vector. J Bacteriol 1990; 172:2793–2797 [View Article][PubMed]
    [Google Scholar]
  37. Davies JF 2nd, Almassy RJ, Hostomska Z, Ferre RA, Hostomsky Z. 2.3 A crystal structure of the catalytic domain of DNA polymerase beta. Cell 1994; 76:1123–1133 [View Article][PubMed]
    [Google Scholar]
  38. De Mot R, Nagy I, De Schrijver A, Pattanapipitpaisal P, Schoofs G et al. Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors. Microbiology 1997; 143:3137–3147 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000447
Loading
/content/journal/micro/10.1099/mic.0.000447
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Most cited Most Cited RSS feed