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. 1995 Jul;177(14):4028–4035. doi: 10.1128/jb.177.14.4028-4035.1995

Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.

A Tabuchi 1, M Ohnishi 1, T Hayashi 1, Y Terawaki 1
PMCID: PMC177133  PMID: 7608076

Abstract

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.

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Selected References

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  1. Abeles A. L., Austin S. J. Antiparallel plasmid-plasmid pairing may control P1 plasmid replication. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9011–9015. doi: 10.1073/pnas.88.20.9011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abeles A. L. P1 plasmid replication. Purification and DNA-binding activity of the replication protein RepA. J Biol Chem. 1986 Mar 15;261(8):3548–3555. [PubMed] [Google Scholar]
  3. Abeles A. L., Reaves L. D., Austin S. J. A single DnaA box is sufficient for initiation from the P1 plasmid origin. J Bacteriol. 1990 Aug;172(8):4386–4391. doi: 10.1128/jb.172.8.4386-4391.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Abeles A. L., Snyder K. M., Chattoraj D. K. P1 plasmid replication: replicon structure. J Mol Biol. 1984 Mar 5;173(3):307–324. doi: 10.1016/0022-2836(84)90123-2. [DOI] [PubMed] [Google Scholar]
  5. Abeles A., Brendler T., Austin S. Evidence of two levels of control of P1 oriR and host oriC replication origins by DNA adenine methylation. J Bacteriol. 1993 Dec;175(24):7801–7807. doi: 10.1128/jb.175.24.7801-7807.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  7. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  8. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  9. Bramhill D., Kornberg A. Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome. Cell. 1988 Mar 11;52(5):743–755. doi: 10.1016/0092-8674(88)90412-6. [DOI] [PubMed] [Google Scholar]
  10. Brendler T., Abeles A., Austin S. Critical sequences in the core of the P1 plasmid replication origin. J Bacteriol. 1991 Jul;173(13):3935–3942. doi: 10.1128/jb.173.13.3935-3942.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chattoraj D. K., Mason R. J., Wickner S. H. Mini-P1 plasmid replication: the autoregulation-sequestration paradox. Cell. 1988 Feb 26;52(4):551–557. doi: 10.1016/0092-8674(88)90468-0. [DOI] [PubMed] [Google Scholar]
  13. Churchward G., Belin D., Nagamine Y. A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene. 1984 Nov;31(1-3):165–171. doi: 10.1016/0378-1119(84)90207-5. [DOI] [PubMed] [Google Scholar]
  14. Coetzee J. N., Datta N., Hedges R. W. R factors from Proteus rettgeri. J Gen Microbiol. 1972 Oct;72(3):543–552. doi: 10.1099/00221287-72-3-543. [DOI] [PubMed] [Google Scholar]
  15. Cohen S. N., Chang A. C., Boyer H. W., Helling R. B. Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3240–3244. doi: 10.1073/pnas.70.11.3240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. DasGupta S., Mukhopadhyay G., Papp P. P., Lewis M. S., Chattoraj D. K. Activation of DNA binding by the monomeric form of the P1 replication initiator RepA by heat shock proteins DnaJ and DnaK. J Mol Biol. 1993 Jul 5;232(1):23–34. doi: 10.1006/jmbi.1993.1367. [DOI] [PubMed] [Google Scholar]
  18. Dempsey L. A., Birch P., Khan S. A. Six amino acids determine the sequence-specific DNA binding and replication specificity of the initiator proteins of the pT181 family. J Biol Chem. 1992 Dec 5;267(34):24538–24543. [PubMed] [Google Scholar]
  19. Easton A. M., Rownd R. H. The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region. J Bacteriol. 1982 Nov;152(2):829–839. doi: 10.1128/jb.152.2.829-839.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Friedman S. A., Austin S. J. The P1 plasmid-partition system synthesizes two essential proteins from an autoregulated operon. Plasmid. 1988 Mar;19(2):103–112. doi: 10.1016/0147-619x(88)90049-2. [DOI] [PubMed] [Google Scholar]
  21. Fuller R. S., Kaguni J. M., Kornberg A. Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7370–7374. doi: 10.1073/pnas.78.12.7370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gibbs M. D., Spiers A. J., Bergquist P. L. RepFIB: a basic replicon of large plasmids. Plasmid. 1993 May;29(3):165–179. doi: 10.1006/plas.1993.1020. [DOI] [PubMed] [Google Scholar]
  23. Gottesman M. E., Yarmolinsky M. B. Integration-negative mutants of bacteriophage lambda. J Mol Biol. 1968 Feb 14;31(3):487–505. doi: 10.1016/0022-2836(68)90423-3. [DOI] [PubMed] [Google Scholar]
  24. Humphreys G. O., Willshaw G. A., Anderson E. S. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim Biophys Acta. 1975 Apr 2;383(4):457–463. doi: 10.1016/0005-2787(75)90318-4. [DOI] [PubMed] [Google Scholar]
  25. Ishiai M., Wada C., Kawasaki Y., Yura T. Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor). Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3839–3843. doi: 10.1073/pnas.91.9.3839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Itoh Y., Kamio Y., Furuta Y., Terawaki Y. Cloning of the replication and incompatibility regions of a plasmid derived from Rts1. Plasmid. 1982 Nov;8(3):232–243. doi: 10.1016/0147-619x(82)90061-0. [DOI] [PubMed] [Google Scholar]
  27. Itoh Y., Kamio Y., Terawaki Y. Essential DNA sequence for the replication of Rts1. J Bacteriol. 1987 Mar;169(3):1153–1160. doi: 10.1128/jb.169.3.1153-1160.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kamio Y., Itoh Y., Terawaki Y. Purification of Rts1 RepA protein and binding of the protein to mini-Rts1 DNA. J Bacteriol. 1988 Sep;170(9):4411–4414. doi: 10.1128/jb.170.9.4411-4414.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kamio Y., Tabuchi A., Itoh Y., Katagiri H., Terawaki Y. Complete nucleotide sequence of mini-Rts1 and its copy mutant. J Bacteriol. 1984 Apr;158(1):307–312. doi: 10.1128/jb.158.1.307-312.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kamio Y., Terawaki Y. Nucleotide sequence of an incompatibility region of mini-Rts1 that contains five direct repeats. J Bacteriol. 1983 Sep;155(3):1185–1191. doi: 10.1128/jb.155.3.1185-1191.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kawasaki Y., Wada C., Yura T. Binding of RepE initiator protein to mini-F DNA origin (ori2). Enhancing effects of repE mutations and DnaJ heat shock protein. J Biol Chem. 1992 Jun 5;267(16):11520–11524. [PubMed] [Google Scholar]
  32. Kawasaki Y., Wada C., Yura T. Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication. Mol Gen Genet. 1990 Jan;220(2):277–282. doi: 10.1007/BF00260494. [DOI] [PubMed] [Google Scholar]
  33. Koop A. H., Hartley M. E., Bourgeois S. A low-copy-number vector utilizing beta-galactosidase for the analysis of gene control elements. Gene. 1987;52(2-3):245–256. doi: 10.1016/0378-1119(87)90051-5. [DOI] [PubMed] [Google Scholar]
  34. Manen D., Upegui-Gonzalez L. C., Caro L. Monomers and dimers of the RepA protein in plasmid pSC101 replication: domains in RepA. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):8923–8927. doi: 10.1073/pnas.89.19.8923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McEachern M. J., Bott M. A., Tooker P. A., Helinski D. R. Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7942–7946. doi: 10.1073/pnas.86.20.7942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Miller J., Manis J., Kline B., Bishop A. Nonintegrated plasmid-folded chromosome complexes: genetic studies on formation and possible relationship to plasmid replication. Plasmid. 1978 Jun;1(3):273–283. doi: 10.1016/0147-619x(78)90045-8. [DOI] [PubMed] [Google Scholar]
  37. Miron A., Patel I., Bastia D. Multiple pathways of copy control of gamma replicon of R6K: mechanisms both dependent on and independent of cooperativity of interaction of tau protein with DNA affect the copy number. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6438–6442. doi: 10.1073/pnas.91.14.6438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mukhopadhyay G., Sozhamannan S., Chattoraj D. K. Relaxation of replication control in chaperone-independent initiator mutants of plasmid P1. EMBO J. 1994 May 1;13(9):2089–2096. doi: 10.1002/j.1460-2075.1994.tb06484.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Murotsu T., Matsubara K., Sugisaki H., Takanami M. Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid. Gene. 1981 Nov;15(2-3):257–271. doi: 10.1016/0378-1119(81)90135-9. [DOI] [PubMed] [Google Scholar]
  40. Nozue H., Tsuchiya K., Kamio Y. Nucleotide sequence and copy control function of the extension of the incI region (incI-b) of Rts 1. Plasmid. 1988 Jan;19(1):46–56. doi: 10.1016/0147-619x(88)90062-5. [DOI] [PubMed] [Google Scholar]
  41. Pal S. K., Chattoraj D. K. P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites. J Bacteriol. 1988 Aug;170(8):3554–3560. doi: 10.1128/jb.170.8.3554-3560.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Projan S. J., Novick R. Comparative analysis of five related Staphylococcal plasmids. Plasmid. 1988 May;19(3):203–221. doi: 10.1016/0147-619x(88)90039-x. [DOI] [PubMed] [Google Scholar]
  43. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Skowyra D., Wickner S. The interplay of the GrpE heat shock protein and Mg2+ in RepA monomerization by DnaJ and DnaK. J Biol Chem. 1993 Dec 5;268(34):25296–25301. [PubMed] [Google Scholar]
  45. Sozhamannan S., Chattoraj D. K. Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin. J Bacteriol. 1993 Jun;175(11):3546–3555. doi: 10.1128/jb.175.11.3546-3555.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Stalker D. M., Kolter R., Helinski D. R. Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1150–1154. doi: 10.1073/pnas.76.3.1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Terawaki Y., Hong Z., Itoh Y., Kamio Y. Importance of the C terminus of plasmid Rts1 RepA protein for replication and incompatibility of the plasmid. J Bacteriol. 1988 Mar;170(3):1261–1267. doi: 10.1128/jb.170.3.1261-1267.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Terawaki Y., Itoh Y., Zeng H., Hayashi T., Tabuchi A. Function of the N-terminal half of RepA in activation of Rts1 ori. J Bacteriol. 1992 Nov;174(21):6904–6910. doi: 10.1128/jb.174.21.6904-6910.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Terawaki Y., Kobayashi Y., Matsumoto H., Kamio Y. An Rts1-derivative plasmid conferring UV sensitivity on Escherichia coli host. Biochem Biophys Res Commun. 1980 Nov 28;97(2):694–699. doi: 10.1016/0006-291x(80)90320-4. [DOI] [PubMed] [Google Scholar]
  50. Terawaki Y., Kobayashi Y., Matsumoto H., Kamio Y. Molecular cloning and mapping of a deletion derivative of the plasmid Rts 1. Plasmid. 1981 Sep;6(2):222–234. doi: 10.1016/0147-619x(81)90068-8. [DOI] [PubMed] [Google Scholar]
  51. Terawaki Y., Nozue H., Zeng H., Hayashi T., Kamio Y., Itoh Y. Effects of mutations in the repA gene of plasmid Rts1 on plasmid replication and autorepressor function. J Bacteriol. 1990 Feb;172(2):786–792. doi: 10.1128/jb.172.2.786-792.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tilly K., Yarmolinsky M. Participation of Escherichia coli heat shock proteins DnaJ, DnaK, and GrpE in P1 plasmid replication. J Bacteriol. 1989 Nov;171(11):6025–6029. doi: 10.1128/jb.171.11.6025-6029.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Vocke C., Bastia D. Primary structure of the essential replicon of the plasmid pSC101. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6557–6561. doi: 10.1073/pnas.80.21.6557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wang P. Z., Projan S. J., Henriquez V., Novick R. P. Origin recognition specificity in pT181 plasmids is determined by a functionally asymmetric palindromic DNA element. EMBO J. 1993 Jan;12(1):45–52. doi: 10.1002/j.1460-2075.1993.tb05630.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wickner S. H. Three Escherichia coli heat shock proteins are required for P1 plasmid DNA replication: formation of an active complex between E. coli DnaJ protein and the P1 initiator protein. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2690–2694. doi: 10.1073/pnas.87.7.2690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wickner S., Hoskins J., McKenney K. Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA. Nature. 1991 Mar 14;350(6314):165–167. doi: 10.1038/350165a0. [DOI] [PubMed] [Google Scholar]
  57. Wickner S., Hoskins J., McKenney K. Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin. Proc Natl Acad Sci U S A. 1991 Sep 15;88(18):7903–7907. doi: 10.1073/pnas.88.18.7903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wickner S., Skowyra D., Hoskins J., McKenney K. DnaJ, DnaK, and GrpE heat shock proteins are required in oriP1 DNA replication solely at the RepA monomerization step. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10345–10349. doi: 10.1073/pnas.89.21.10345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  60. York D., Filutowicz M. Autoregulation-deficient mutant of the plasmid R6K-encoded pi protein distinguishes between palindromic and nonpalindromic binding sites. J Biol Chem. 1993 Oct 15;268(29):21854–21861. [PubMed] [Google Scholar]
  61. Zeng H., Hayashi T., Terawaki Y. Site-directed mutations in the repA C-terminal region of plasmid Rts1: pleiotropic effects on the replication and autorepressor functions. J Bacteriol. 1990 May;172(5):2535–2540. doi: 10.1128/jb.172.5.2535-2540.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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