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EMBO J. Aug 3, 1998; 17(15): 4511–4526.
PMCID: PMC1170782

Protein domains and conformational changes in the activation of RepA, a DNA replication initiator.


RepA is the DNA replication initiator protein of the Pseudomonas plasmid pPS10. RepA has a dual function: as a dimer, it binds to an inversely-repeated sequence acting as a repressor of its own synthesis; as a monomer, RepA binds to four directly-repeated sequences to constitute a specialized nucleoprotein complex responsible for the initiation of DNA replication. We have previously shown that a Leucine Zipper-like motif (LZ) at the N-terminus of RepA is responsible for protein dimerization. In this paper we characterize the existence in RepA of two protein globular domains C-terminal to the LZ. We propose that dissociation of RepA dimers into monomers results in a conformational change from a compact arrangement of both domains, competent for binding to the operator, to an extended species that is suited for iteron binding. This model establishes the structural basis for the activation of DNA replication initiators in plasmids from Gram-negative bacteria.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ackers GK. Analytical gel chromatography of proteins. Adv Protein Chem. 1970;24:343–446. [PubMed]
  • Andrade MA, Chacón P, Merelo JJ, Morán F. Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng. 1993 Jun;6(4):383–390. [PubMed]
  • Bell SP, Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992 May 14;357(6374):128–134. [PubMed]
  • Brennan RG. The winged-helix DNA-binding motif: another helix-turn-helix takeoff. Cell. 1993 Sep 10;74(5):773–776. [PubMed]
  • Brennan RG, Matthews BW. The helix-turn-helix DNA binding motif. J Biol Chem. 1989 Feb 5;264(4):1903–1906. [PubMed]
  • Charnock C. Characterization of the cryptic plasmids of the Pseudomonas alcaligenes type strain. Plasmid. 1997;37(3):189–198. [PubMed]
  • Chattoraj DK, Schneider TD. Replication control of plasmid P1 and its host chromosome: the common ground. Prog Nucleic Acid Res Mol Biol. 1997;57:145–186. [PubMed]
  • Chattoraj DK, Ghirlando R, Park K, Dibbens JA, Lewis MS. Dissociation kinetics of RepA dimers: implications for mechanisms of activation of DNA binding by chaperones. Genes Cells. 1996 Feb;1(2):189–199. [PubMed]
  • Corbett RJ, Roche RS. Use of high-speed size-exclusion chromatography for the study of protein folding and stability. Biochemistry. 1984 Apr 10;23(8):1888–1894. [PubMed]
  • DasGupta S, Mukhopadhyay G, Papp PP, Lewis MS, Chattoraj DK. 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. [PubMed]
  • Dean FB, O'Donnell M. Two steps to binding replication origins? DNA-protein interactions. Curr Biol. 1996 Aug 1;6(8):931–934. [PubMed]
  • del Solar G, Giraldo R, Ruiz-Echevarría MJ, Espinosa M, Díaz-Orejas R. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. 1998 Jun;62(2):434–464. [PMC free article] [PubMed]
  • Dibbens JA, Muraiso K, Chattoraj DK. Chaperone-mediated reduction of RepA dimerization is associated with RepA conformational change. Mol Microbiol. 1997 Oct;26(1):185–195. [PubMed]
  • Dodson M, Roberts J, McMacken R, Echols H. Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: complexes with lambda O protein and with lambda O, lambda P, and Escherichia coli DnaB proteins. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4678–4682. [PMC free article] [PubMed]
  • Fernández-Tresguerres ME, Martín M, García de Viedma D, Giraldo R, Díaz-Orejas R. Host growth temperature and a conservative amino acid substitution in the replication protein of pPS10 influence plasmid host range. J Bacteriol. 1995 Aug;177(15):4377–4384. [PMC free article] [PubMed]
  • Filutowicz M, Davis G, Greener A, Helinski DR. Autorepressor properties of the pi-initiation protein encoded by plasmid R6K. Nucleic Acids Res. 1985 Jan 11;13(1):103–114. [PMC free article] [PubMed]
  • Filutowicz M, York D, Levchenko I. Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution. Nucleic Acids Res. 1994 Oct 11;22(20):4211–4215. [PMC free article] [PubMed]
  • García de Viedma D, Giraldo R, Ruiz-Echevarría MJ, Lurz R, Díaz-Orejas R. Transcription of repA, the gene of the initiation protein of the Pseudomonas plasmid pPS10, is autoregulated by interactions of the RepA protein at a symmetrical operator. J Mol Biol. 1995 Mar 24;247(2):211–223. [PubMed]
  • García de Viedma D, Serrano-López A, Díaz-Orejas R. Specific binding of the replication protein of plasmid pPS10 to direct and inverted repeats is mediated by an HTH motif. Nucleic Acids Res. 1995 Dec 25;23(24):5048–5054. [PMC free article] [PubMed]
  • Garcia de Viedma D, Giraldo R, Rivas G, Fernández-Tresguerres E, Diaz-Orejas R. A leucine zipper motif determines different functions in a DNA replication protein. EMBO J. 1996 Feb 15;15(4):925–934. [PMC free article] [PubMed]
  • Giraldo R, Díaz R. Differential binding of wild-type and a mutant RepA protein to oriR sequence suggests a model for the initiation of plasmid R1 replication. J Mol Biol. 1992 Dec 5;228(3):787–802. [PubMed]
  • Giraldo R, Nieto C, Fernandez-Tresguerres ME, Diaz R. Bacterial zipper. Nature. 1989 Dec 21;342(6252):866–866. [PubMed]
  • Gottesman S. Minimizing proteolysis in Escherichia coli: genetic solutions. Methods Enzymol. 1990;185:119–129. [PubMed]
  • Gottesman S, Wickner S, Maurizi MR. Protein quality control: triage by chaperones and proteases. Genes Dev. 1997 Apr 1;11(7):815–823. [PubMed]
  • Horii T, Ogawa T, Ogawa H. Organization of the recA gene of Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jan;77(1):313–317. [PMC free article] [PubMed]
  • Hubbard SJ. The structural aspects of limited proteolysis of native proteins. Biochim Biophys Acta. 1998 Feb 17;1382(2):191–206. [PubMed]
  • Hwang DS, Crooke E, Kornberg A. Aggregated dnaA protein is dissociated and activated for DNA replication by phospholipase or dnaK protein. J Biol Chem. 1990 Nov 5;265(31):19244–19248. [PubMed]
  • Ingmer H, Fong EL, Cohen SN. Monomer-dimer equilibrium of the pSC101 RepA protein. J Mol Biol. 1995 Jul 14;250(3):309–314. [PubMed]
  • 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Kawasaki Y, Wada C, Yura T. Mini-F plasmid mutants able to replicate in the absence of sigma 32: mutations in the repE coding region producing hyperactive initiator protein. J Bacteriol. 1991 Feb;173(3):1064–1072. [PMC free article] [PubMed]
  • Kelly SM, Price NC. The application of circular dichroism to studies of protein folding and unfolding. Biochim Biophys Acta. 1997 Apr 4;1338(2):161–185. [PubMed]
  • Kelman Z, O'Donnell M. DNA replication: enzymology and mechanisms. Curr Opin Genet Dev. 1994 Apr;4(2):185–195. [PubMed]
  • Konieczny I, Helinski DR. The replication initiation protein of the broad-host-range plasmid RK2 is activated by the ClpX chaperone. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14378–14382. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lee DG, Bell SP. Architecture of the yeast origin recognition complex bound to origins of DNA replication. Mol Cell Biol. 1997 Dec;17(12):7159–7168. [PMC free article] [PubMed]
  • Lefstin JA, Yamamoto KR. Allosteric effects of DNA on transcriptional regulators. Nature. 1998 Apr 30;392(6679):885–888. [PubMed]
  • Levchenko I, York D, Filutowicz M. The dimerization domain of R6K plasmid replication initiator protein pi revealed by analysis of a truncated protein. Gene. 1994 Jul 22;145(1):65–68. [PubMed]
  • Levchenko I, Inman RB, Filutowicz M. Replication of the R6K gamma origin in vitro: dependence on wt pi and hyperactive piS87N protein variant. Gene. 1997 Jul 1;193(1):97–103. [PubMed]
  • Lupas A. Coiled coils: new structures and new functions. Trends Biochem Sci. 1996 Oct;21(10):375–382. [PubMed]
  • Manen D, Upegui-Gonzalez LC, 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. [PMC free article] [PubMed]
  • Matsunaga F, Ishiai M, Kobayashi G, Uga H, Yura T, Wada C. The central region of RepE initiator protein of mini-F plasmid plays a crucial role in dimerization required for negative replication control. J Mol Biol. 1997 Nov 21;274(1):27–38. [PubMed]
  • Miron A, Mukherjee S, Bastia D. Activation of distant replication origins in vivo by DNA looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance. EMBO J. 1992 Mar;11(3):1205–1216. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Mukhopadhyay G, Sozhamannan S, Chattoraj DK. Relaxation of replication control in chaperone-independent initiator mutants of plasmid P1. EMBO J. 1994 May 1;13(9):2089–2096. [PMC free article] [PubMed]
  • Newlon CS. Putting it all together: building a prereplicative complex. Cell. 1997 Dec 12;91(6):717–720. [PubMed]
  • Nieto C, Giraldo R, Fernández-Tresguerres E, Díaz R. Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae patovar savastanoi. J Mol Biol. 1992 Jan 20;223(2):415–426. [PubMed]
  • Nozaki Y, Schechter NM, Reynolds JA, Tanford C. Use of gel chromatography for the determination of the Stokes radii of proteins in the presence and absence of detergents. A reexamination. Biochemistry. 1976 Aug 24;15(17):3884–3890. [PubMed]
  • Olins PO, Devine CS, Rangwala SH, Kavka KS. The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli. Gene. 1988 Dec 15;73(1):227–235. [PubMed]
  • Pak M, Wickner S. Mechanism of protein remodeling by ClpA chaperone. Proc Natl Acad Sci U S A. 1997 May 13;94(10):4901–4906. [PMC free article] [PubMed]
  • Potschka M. Universal calibration of gel permeation chromatography and determination of molecular shape in solution. Anal Biochem. 1987 Apr;162(1):47–64. [PubMed]
  • Rokeach LA, Søgaard-Andersen L, Molin S. Two functions of the E protein are key elements in the plasmid F replication control system. J Bacteriol. 1985 Dec;164(3):1262–1270. [PMC free article] [PubMed]
  • Rost B, Sander C. Prediction of protein secondary structure at better than 70% accuracy. J Mol Biol. 1993 Jul 20;232(2):584–599. [PubMed]
  • Semisotnov GV, Rodionova NA, Razgulyaev OI, Uversky VN, Gripas' AF, Gilmanshin RI. Study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers. 1991 Jan;31(1):119–128. [PubMed]
  • Soisson SM, MacDougall-Shackleton B, Schleif R, Wolberger C. Structural basis for ligand-regulated oligomerization of AraC. Science. 1997 Apr 18;276(5311):421–425. [PubMed]
  • Sozhamannan S, Chattoraj DK. 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. [PMC free article] [PubMed]
  • Vocke C, Bastia D. The replication initiator protein of plasmid pSC101 is a transcriptional repressor of its own cistron. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2252–2256. [PMC free article] [PubMed]
  • Wemmer DE, Dervan PB. Targeting the minor groove of DNA. Curr Opin Struct Biol. 1997 Jun;7(3):355–361. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Wickner S, Gottesman S, Skowyra D, Hoskins J, McKenney K, Maurizi MR. A molecular chaperone, ClpA, functions like DnaK and DnaJ. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12218–12222. [PMC free article] [PubMed]
  • Wu J, Sektas M, Chen D, Filutowicz M. Two forms of replication initiator protein: positive and negative controls. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13967–13972. [PMC free article] [PubMed]
  • Xia G, Manen D, Yu Y, Caro L. In vivo and in vitro studies of a copy number mutation of the RepA replication protein of plasmid pSC101. J Bacteriol. 1993 Jul;175(13):4165–4175. [PMC free article] [PubMed]
  • 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. [PubMed]
  • 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]

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