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Journal of Bacteriology, February 2007, p. 1061-1071, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.01455-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Specific Interaction between the Initiator Protein (Rep) and Origin of Plasmid ColE2-P9{triangledown}

M. Han, M. Yagura, and T. Itoh*

Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano 390-8621, Japan

Received 14 September 2006/ Accepted 1 November 2006


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ABSTRACT
 
The replication initiator protein (Rep) of plasmid ColE2-P9 (ColE2) is multifunctional. We are interested in how Rep binds to the origin (Ori) to perform various functions. We used the wild type and variants of Rep to study the Rep-Ori interaction by both in vitro and in vivo approaches, including biochemical analyses of protein-DNA interactions and an in vivo replication assay. We identified three regions (I, II, and III) of Rep, located in the C-terminal half, and three corresponding binding sites (I, II, and III) in Ori which are important for Rep-Ori interaction. We showed that region I, containing a putative helix-turn-helix motif, is necessary and sufficient for specific Ori recognition, interacting with site I of the origin DNA from the major groove. Region II interacts with site II of the origin DNA, from the adjacent minor groove in the left half of Ori, and region III interacts with site III, next to the template sequence for primer synthesis, which is one and one-half turn apart from site I on the opposite surface of the origin DNA. A putative linker region located between the two DNA binding domains (regions II and III) was identified, which might provide Rep an extended conformation suitable for binding to the two separate sites in Ori. Based on the results presented in this paper, we propose a model for Rep-Ori interaction in which Rep binds to Ori as a monomer.


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INTRODUCTION
 
DNA replication is a universal process for transmission of genetic information in all organisms. In the classic replicon model (14), initiation of DNA replication requires a cis-acting DNA sequence (the replicator) and a trans-acting factor (the initiator) that binds to the replicator, which is now called the origin (of replication). The origin-bound initiators melt the duplex DNA strands in a region adjacent to the origin to form an open complex (5, 9, 20). The initiators then introduce other replication proteins, such as the helicase, primase, and DNA polymerase, into the open complex to form the replisome. Among various initiators, the DnaA protein of Escherichia coli is the best characterized (15). Its ATP-bound form binds as a monomer specifically to each of the four asymmetric 9-bp consensus sequences (5'-TTA/TTNCACA) or to closely related sequences, the DnaA boxes in the origin (OriC). These ATP-bound DnaA proteins oligomerize with additional ATP-bound DnaA proteins to form a large nucleoprotein structure containing 20 to 40 DnaA proteins, which induces local unwinding of the AT-rich region adjacent to OriC. The DnaA proteins act as a replisome organizer for the subsequent loading of proteins required for the replication process (23).

In most plasmid replicons, initiation of plasmid replication requires plasmid-encoded initiator proteins, called Rep proteins (5). The crystal structure of the RepE initiator protein of plasmid F was solved as the first three-dimensional structure of a prokaryotic initiator protein (19). The RepE protein consists of topologically similar N- and C-terminal domains related to each other by internal pseudo-twofold symmetry. By using helix-turn-helix (HTH) DNA binding motifs, the two domains bind to two consecutive major grooves on one face of the DNA helix of each of the 19-bp directly repeated sequences (the iterons) in the origin. The amino acid sequence homology between the RepE protein and some other plasmid initiator proteins, such as those of pPS10 and pSC101, suggested that those proteins might bind to their iterons similarly (19). Although the P1 RepA protein cannot be aligned due to its poor sequence homology with the RepE protein, molecular modeling suggested that it is also structurally similar to the class of plasmid initiator proteins to which the RepE protein belongs (28). The replication initiation of these iteron plasmids involves binding of the plasmid-encoded initiator proteins to the iterons and formation of an open complex at their cognate origins to recruit the replisome (5, 16).

The plasmid ColE2-P9 (ColE2) is a circular duplex DNA molecule of about 7 kb (10). It is present at about 10 to 15 copies per host chromosome (1, 12). The initiator protein (Rep; 35 kDa) of plasmid ColE2 is the only plasmid-specified trans-acting factor required for the initiation of plasmid replication (17, 18, 37). Initiation of plasmid replication also requires host DNA polymerase I (13, 31), but not RNA polymerase and DnaG primase (13, 32). The minimal cis-acting region, or origin (Ori) (Fig. 1A), required for ColE2 DNA replication consists of 31 bp (35). The ColE2 Rep protein binds specifically to Ori DNA, as revealed by a filter binding assay (17) and by an electrophoresis mobility shift assay (EMSA) (35). In an in vitro ColE2 DNA replication system with crude extracts of Escherichia coli, ColE2 DNA replication starts at a unique site in Ori and proceeds unidirectionally (13, 32). The ColE2 Rep protein is unique among other initiator proteins in that it is also a plasmid-specific primase. It synthesizes a three-nucleotide primer RNA which has a unique structure of 5' ppApGpA (33). Host DNA polymerase I specifically uses the primer RNA to start DNA synthesis and then form a D-loop structure, into which various replication proteins of E. coli, such as DnaB helicase, DnaG primase, and the DNA polymerase III holoenzyme, are introduced to continue replication of ColE2 DNA.


Figure 1
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  		FIG. 1. Origin and Rep protein of plasmid ColE2. (A) Nucleotide sequence of the origin of plasmid ColE2-P9. Positions are numbered from left to right. Sites {alpha} and ß (indicated by arrows below the sequences) are the specificity determinants of the Rep-Ori interaction between ColE2 and ColE3 (31, 37). Subregions I, II, and III of the 31-bp minimal region were identified by mutation analyses (37), and sites a, b, and c were proposed as the Rep protein binding elements, as indicated below the sequence. Sites I, II, and III of the ColE2 origin bound by Rep, identified by DMS protection assays in this study, are boxed. The positions of the ends of deletions from the right (dr8, dr4, and dr56) (Table 2) are indicated by bent arrows. (B) Schematic representations of the wild type and partial fragments of Rep (solid bars) used in this study. Rep fragments with deletions from the N terminus were named with the numbers of the remaining residues in the C-terminal region. Rep{Delta}C20 is a stop codon mutant Rep protein lacking 20 residues in the C-terminal region. The position numbers of the residues at the N and C termini are indicated. (C) C-terminal 147-amino-acid region of the ColE2 Rep protein important for binding to origin DNA. The putative HTH motif showing sequence homology with E. coli transcription factors (13) and regions A and B, involved in determining the specificity of the Rep-Ori interaction in plasmids ColE2-P9 and ColE3-CA38 (31), are indicated by double-headed arrows below and above the sequence, respectively. The positions of the deletion ends of the Rep fragments are indicated by bent arrows. The single amino acid substitutions in mutant Rep proteins which retained or lost the in vivo ability to initiate DNA replication are indicated above and below the sequence, respectively. The predicted {alpha} helix regions (Psi-Pred) are indicated by solid bars below the sequence. Regions I, II, and III of the Rep protein, identified in this study as important for binding to the origin DNA, are boxed, and a putative linker region is indicated by a bracket above the sequence.

The ColE2 Rep protein has a potential HTH DNA binding motif, based on amino acid sequence homology to some known E. coli DNA binding proteins (11). Comparative studies on the replication of plasmid ColE2 and its close relative, plasmid ColE3-CA38, have shown that interactions of the Rep proteins with the origins of these two plasmids are plasmid specific, although both of their Rep proteins and origins show high degrees of similarity. By using chimeric rep genes and chimeric origins, the two regions (A and B) in the C-terminal regions of the Rep proteins and the two sites ({alpha} and ß) in the origins important for the determination of plasmid specificity were identified (Fig. 1A and C) (29). Region B in the C-terminal region of Rep, corresponding to the second recognition helix of a potential HTH DNA binding motif (11), was proposed to be a part of the sequence-specific DNA binding domain (DBD), and region A might be a linker connecting two domains in Rep involved in DNA binding (29).

Mutational analyses (35) have revealed that Ori may be divided into three subregions (I, II, and III). Subregion I is important for stable binding of Rep and contains site ß. Subregion II is important for binding of Rep and for initiation of DNA replication and contains site {alpha}. Subregion III is important for DNA replication but not, apparently, for binding of Rep and contains the site where the primer is synthesized. It has also been suggested that there are three Rep binding elements in Ori, with one (site a) in subregion I and two (sites b and c) in subregion II.

Detailed analysis of the binding properties of ColE2 Rep and Ori is an important prerequisite to investigating the following steps after Rep binding to Ori, such as the formation of the open complex and primer synthesis, providing insights into the mechanism of initiation of ColE2 DNA replication. In this study, we used EMSA and a dimethyl sulfate (DMS) protection assay to characterize the interaction of Rep and Ori. We identified three regions in Rep involved in Ori binding and three corresponding binding sites in Ori. We propose a model for the mode of ColE2 Rep-Ori binding.


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MATERIALS AND METHODS
 
Strains and plasmids. The E. coli strains used were AG1 recA1 (11) and BL21(DE3) (Novagen). The plasmids used have been described elsewhere (3, 12, 24, 29, 35, 37), except for those described below.

Construction of plasmids expressing Rep fragments. The plasmid pET21a-E2Rep+site (35) is a derivative of pET21a(+) (Novagen) carrying the ColE2 rep gene, in which two new endonuclease cleavage sites (SacII and SalI) were generated without any changes in the encoded amino acid residues. Plasmid pACYC177 (3) was digested with DraI, followed by self-ligation, to construct pACYC177dAp, in which the ß-lactamase gene was inactivated. To construct pHME2Rep+Km, the entire pACYC177dAp plasmid cleaved with BstBI was inserted at the BstBI site of pET21a-E2Rep+site, located in the region encoding the C-terminal one-third of the Rep protein. To construct pHMC132, the 4.12-kb EcoRI (filled with T4 DNA polymerase)-BglII fragment of pHME2Rep+Km, containing pACYC177dAp and portions of the ColE2 rep gene, was replaced with PCR fragments (amplified with oligonucleotides KM3F and KM3R) digested with the BglII enzyme. The construction of pHMC94, pHMC62, and pHMC37 was the same as that described for pHMC132, except for the oligonucleotides used for amplification of the PCR fragments (d203 and KM3R for pHMC94, d236 and KM3R for pHMC62, and d260 and KM3R for pHMC37). Plasmid pHMC274 was constructed by digesting pETa21-E2Rep+site with EcoRI and SplI, followed by filling with T4 DNA polymerase and self-ligation. To construct pHMC169, the BamHI (filled with T4 DNA polymerase)-BglII fragment of pHME2Rep+Km, containing pACYC177dAp and portions of the ColE2 rep gene, was replaced with a PCR fragment (amplified with oligonucleotides KM2F and KM2R) digested with MboI (filled with T4 DNA polymerase) and Bsp119I. To construct pETE2RepC117 (K. Matsumoto and T. Itoh, unpublished data), pET21a-E2Rep+site was digested with EcoRI and AsuII, followed by filling with the Klenow fragment and self-ligation. To construct pHMC45, the EcoRI (filled with T4 DNA polymerase)-BglII fragment of pHME2Rep+Km, containing pACYC177dAp and portions of the ColE2 rep gene, was replaced with a PCR fragment (amplified with oligonucleotides KM3F and KM3R) digested with XhoI (filled with T4 DNA polymerase) and BglII.

Construction of plasmids expressing mutant Rep proteins with single amino acid substitutions. To introduce single amino acid substitutions in the C-terminal half of the Rep protein, PCR in the presence of MnCl2 was performed in a reaction mixture (20 µl) containing 0.02 µg of the template DNA (pETa21-E2Rep+site), 10 pmol each of oligonucleotides KM3F and KM3R, 0.5 units Gene Taq DNA polymerase (Wako), 1 µl of 10x Gene Taq buffer, a 0.2 mM concentration of each deoxynucleoside triphosphate, and 0.35 mM MnCl2. Amplification was done through 30 cycles of denaturation (94°C for 1 min), annealing (55°C for 40 s), and polymerization (72°C for 30 s). PCR fragments digested with SalI and BglII were inserted between the SalI and BglII sites of pHME2Rep+Km, and the nucleotide sequences of the PCR-amplified portions of the resultant plasmids were determined to identify single amino acid substitutions in the Rep proteins encoded by the mutant plasmids. In some cases, in vivo replication activities (see below) of the resultant plasmids were measured before sequencing to screen the mutant Rep proteins defective in replication. The plasmid carrying the mutant rep gene encoding the Rep{Delta}C20 protein was among them. The mutagenic oligonucleotide KA1 (Table 1) and oligonucleotide KM3F were used to construct plasmids producing the mutant Rep proteins RepT284W and RepR287Q.


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  		TABLE 1. Oligonucleotides used for mutagenesis

Construction of other plasmids. The 1.5-kb BamHI-BglII fragment of pEC22X43 (12), containing the ColE2 rep gene with an XbaI linker inserted in the RNA I coding region, and the 2.1-kb BamHI-BglII fragment of pTI20 (37), containing the ColE1 replicon and the ß-lactamase gene, were ligated to construct pTI243+. The 2.4-kb EcoRI-DraI fragment of pTI243+, containing the ColE1 replicon and the ColE2 rep gene, and the 1.4-kb EcoRI-SmaI segment of pKC7 (24), containing the kanamycin resistance gene, were ligated to construct pTIK243+. The 252-bp EcoRI-EcoRV fragment of pTI51dr8 (35) and the 258-bp EcoRI-EcoRV fragment of pTI51dr4 (35) were cloned between the EcoRI and EcoRV sites of pBluescript KS(+), followed by digestion with NheI and XhoI and by self-ligation to construct pHMdr8 and pHMdr4, respectively. The 419-bp EcoRI-HindIII fragment of pTI51dr56 (35) was cloned between the EcoRI-HindIII sites of pBluescript KS(+) and then digested with NheI and XhoI, followed by self-ligation, to construct pHM dr56.

Media, enzymes, and chemicals. The media used have been described elsewhere (12). Chemicals, enzymes, and antibiotics used were from commercial sources. [{alpha}-32P]dCTP (3,000 Ci/mmol) was obtained from Amersham Biosciences. The oligonucleotides used for mutagenesis were obtained from Pharmacia and are listed in Table 1.

In vivo replication assay. Derivatives of pETa21-E2Rep+site producing the mutant Rep proteins with single amino acid substitutions or pETa21-E2Rep+site (as a positive control) was introduced into E. coli AG1 cells carrying pTIK243+ and pEC22s (29), containing the chloramphenicol resistance gene and the ColE2 origin. Cells were plated onto an LB agar plate containing ampicillin (50 µg/ml). After incubation for 14 h at 37°C, 20 randomly selected colonies were transferred to a new LB agar plate containing ampicillin and incubated for 14 h at 37°C. Cells grown from each colony were transferred to three new LB agar plates, containing ampicillin (50 µg/ml) and kanamycin (20 µg/ml), ampicillin (50 µg/ml) and chloramphenicol (20 µg/ml), and ampicillin only, and then incubated for 14 h at 37°C. During the incubation with ampicillin only, pTIK243+ (containing a ColE1 replicon) was excluded from cells by pET21a+E2Rep+site (containing a pBR322 replicon) or its derivatives due to unidirectional incompatibility (8, 34). Growth or no growth of cells on the plates containing ampicillin and chloramphenicol indicates whether the mutant Rep proteins produced by pET21a+E2Rep+site derivatives retain replication activity or not.

Purification of Rep protein. The ColE2 Rep protein, its fragments, and mutant Rep proteins with His6 tags at their C termini were purified from BL21(DE3) cells carrying derivatives of pET21a(+) producing these peptides essentially as described previously (35), except for the step of expression of the peptides. Cells were grown for 3 h at 37°C in 2 ml of Terrific broth (32) supplemented with 50 µg/ml ampicillin. The culture was diluted to 1 x 107 to 2 x 107 cells/ml with 50 ml fresh medium, grown at 25°C to 1 x 108 cells/ml, and treated with 0.4 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) for another 4 h. The protein concentration was determined by the bicinchoninic acid method (30). Bovine serum albumin was used as a standard. The amount of overexpressed wild-type Rep protein in the whole-cell lysate was about 20% of the total E. coli proteins, and the amount of soluble Rep protein in the supernatant after high-speed centrifugation was about 10 to 20% of the total Rep protein. Most of the Rep fragments showed better solubility than intact Rep, and >50% of the total amount was in the supernatant fractions, except for RepC274 and RepC94, whose solubilities were similar to and less than (about 10%) that of the wild-type Rep protein, respectively. The solubilities of the overexpressed mutant Rep proteins were approximately similar to that of the wild-type Rep protein.

EMSA. The origin-specific DNA probe used in the electrophoresis mobility shift assay was generated by digesting pBlue22+wtori (35) with BssHII and 3' end labeling it with [{alpha}-32P]dCTP and the Klenow fragment at room temperature for 30 min. The 230-bp labeled BssHII fragment, containing the ColE2 origin, and an 83-bp BssHII fragment without the ColE2 origin (as a negative control) were purified by use of a PCR purification kit (QIAGEN). The 5 nM labeled origin DNA fragment was incubated for 15 min at room temperature with the wild-type or mutant Rep proteins (up to 500 nM) in 27-µl reaction mixtures containing the binding buffer (200 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM KCl, 0.2 mM EDTA, 10% ethylene glycol), 40 µg/ml bovine serum albumin, and 20 µg/ml salmon DNA. Aliquots were loaded into 8% polyacrylamide-0.5x Tris-borate-EDTA gels (20 x 20 cm2). Electrophoresis was run at 4°C at 420 V (constant voltage). DNAs were visualized by using a Fuji BAS1500 phosphorimager and Image Reader v. 1.7J software.

Western blot analysis. BL21(DE3) cells carrying derivatives of pET21a+E2Rep+site producing mutant Rep proteins with single amino acid substitutions were grown to stationary phase in the absence of IPTG and washed in buffer (1 mM Na2HPO4, 1 mM NaH2PO4, 1 M NaCl, 20 mM imidazole, and 0.5% Tween 20). Proteins in the whole-cell extracts were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by staining with Coomassie brilliant blue. Samples for Western blotting were separated in parallel in another 15% SDS-PAGE gel, and proteins were transferred to a polyvinylidene difluoride sheet (Atto). The sheet was treated with the anti-T7 tag antibody (Novagen) at a 1:8,000 dilution for 1 h and then with nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) (Roche) at room temperature as recommended by the supplier (Novagen manual).

Dimethyl sulfate footprinting on supercoiled plasmid. The wild-type or mutant Rep proteins (up to 400 nM) were allowed to bind to 0.2 pmol supercoiled DNA of pBlue22+wtori in the binding buffer (54 µl) used for EMSA for 10 min at room temperature. Modification reactions were started by precisely adding 1.1 µl of pure DMS (Sigma) at a final concentration of 200 mM, and the reaction mixes were incubated at room temperature. Reactions were stopped after exactly 3 min by adding 110 µl stop solution (1 M ß-mercaptoethanol, 20 mM EDTA), and DMS was thoroughly eliminated by ethanol precipitation.

Primer extension. Modified plasmid DNA molecules treated with DMS were redissolved in 15 µl of distilled water (milliQ). Primer extension was performed in a 10-µl reaction mixture containing 0.1 pmol plasmid DNA, 0.5 units of EX Taq (TAKARA), 1 µl of 10x EX Taq buffer, a 0.25 mM concentration of each deoxynucleoside triphosphate, and 1 pmol each of fluorescein isothiocyanate (FITC)-labeled M13 reverse primer (5'-FITC-GGAAACAGCTATGACCA-3') and M13-20 primer (5'-FITC-GTAAAACGACGGCCAGT-3'). Amplification was done through 36 cycles of denaturation (95°C for 1 min), annealing (50°C for 1 min), and polymerization (72°C for 1 min). The DNA molecules were purified by ethanol precipitation, dissolved in 2 µl of loading dye solution, and heated at 90°C for 2 min. Samples were loaded into an 8% polyacrylamide-urea sequencing gel and run in 1.2x TBE buffer (108 mM Tris-HCl, 2.4 mM EDTA-2 Na, 108 mM boric acid, pH 8.3) for 8 h (DSQ 1000 DNA sequencer; Shimadzu). The nucleotides modified by DMS were precisely localized by comparison with dideoxy sequencing ladders obtained with unmodified pBlue22+wtori plasmid, using the same primer as that used for primer extension and running the reactions side by side.


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RESULTS
 
Analysis of specific Rep-Ori binding by EMSA. In order to identify the regions in Rep important for Ori recognition and binding, we constructed a series of partial Rep fragments lacking either the N-terminal portion or the C-terminal portion and mutant Rep proteins with single amino acid substitutions (Fig. 1B and C). The selection of end points in partial Rep fragments was made with reference to the results of secondary structure prediction (Psi-Pred) for Rep (Fig. 1C).

We then compared the Ori binding activities of the intact Rep protein and its partial fragments by EMSA (Fig. 2A). The wild-type Rep protein bound specifically to the Ori sequence, as reported previously (35). All of the Rep fragments, including RepC117 (Matsumoto and Itoh, unpublished data), also bound specifically to Ori. This indicated that they folded properly to form the sequence-specific DBDs. Note that detailed comparison of the binding affinities of the intact Rep protein and the partial fragments is difficult, as we do not know the ratios of the active forms of these proteins in the soluble fractions. RepC37, consisting of the C-terminal 37 amino acids of the Rep protein with a putative HTH DNA binding motif (positions 261 to 297) (Fig. 1C), bound to Ori specifically (Fig. 2A). This suggested that the putative HTH motif alone is probably sufficient for the sequence-specific binding to Ori.


Figure 2
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  		FIG. 2. Specific Ori DNA binding of ColE2 Rep protein, its partial fragments, and mutant Rep proteins analyzed by EMSA. Various concentrations of peptides, as indicated at the top of each panel, were incubated with end-labeled DNA fragments with (Ori DNA) or without (Ori-free DNA) the origin sequence, and the DNA-protein complexes and free DNA fragments were analyzed by electrophoresis on an 8% polyacrylamide gel. Partial Rep fragments with deletions from the N terminus (A; the positions of DNA-protein complexes are indicated by black circles), Rep{Delta}C20 (B; crude extracts containing this protein was used), a mutant Rep protein with a substitution at position 285 (C), and mutant Rep proteins with substitutions at positions 284, 286, and 287 (D) were compared with the wild-type Rep protein. Additional shifted bands with faster mobilities were occasionally observed depending on the preparations of the Rep proteins used. They were observed more often with proteins of less purity.

To show further the importance of the putative HTH motif in the specific recognition of Ori, we used a deletion mutant Rep protein named Rep{Delta}C20 lacking the C-terminal 20-amino-acid region of Rep containing the second recognition helix of the putative HTH motif (Fig. 1C). It failed to form a complex with Ori DNA (Fig. 2B), indicating that it cannot bind to Ori specifically. This result suggested that the second helix of the putative HTH motif is critical to specific Ori DNA recognition. We further used four mutant Rep proteins with single amino acid substitutions in the second helix of the putative HTH motif, at positions 284, 285, 286, and 287 (Fig. 1C), and examined their Ori binding activities by EMSA (Fig. 2C and D). All of them were defective, showing the importance of these amino acids. All of these results also suggested that the second helix of the putative HTH motif is critical to specific Ori DNA recognition and that these four amino acid residues may be involved directly in the specific recognition. All of these mutant Rep proteins and Rep{Delta}C20 lost the in vivo ability to initiate DNA replication at Ori (measured by the method described in Materials and Methods).

Substitutions or deletion of amino acids at the C-terminal region might have reduced or abolished the stability of the mutant Rep proteins. To exclude this possibility, the expression of the T7-tagged wild-type and mutant Rep proteins in uninduced E. coli cells was detected by using anti-T7 tag antibody (Fig. 3B), as the Rep proteins were invisible by dye staining (Fig. 3A). The amounts of the mutant Rep proteins in uninduced E. coli extracts were approximately similar to that of wild-type Rep. This suggested that all of the mutant Rep proteins were as stable as the wild-type Rep protein and that the defects of the mutant Rep proteins in initiation of in vivo DNA replication were not due to the absence of these proteins in vivo.


Figure 3
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  		FIG. 3. Amounts of ColE2 Rep protein and mutant Rep proteins with a T7 tag in uninduced BL21(DE3) cells carrying plasmids with the cloned genes encoding these proteins. (A) Total E. coli proteins in BL21(DE3) cells were analyzed by 15% SDS-PAGE. (B) Half the amount of each sample used in panel A was loaded in an SDS-PAGE gel, and Western blot analysis was carried out with anti-T7 tag antibody.

DMS protection assay to identify Rep contact sites in Ori and additional domains in Rep involved in Ori binding. Since it is difficult to examine detailed properties of the Rep-Ori interaction and to identify other DNA binding domains in Rep besides the putative HTH motif in the C-terminal region by using EMSA, we performed DMS protection assays for further analyses. DMS methylates the N-7 position of guanine from the major groove and the N-3 position of adenine from the minor groove on double-stranded DNA (dsDNA) substrates (21). Adenine residues are less reactive to DMS than are guanine residues (and therefore the intensities of signals at A residues in the DMS footprints are usually lower than those at G residues). On ssDNA, DMS modifies the N-1 position of adenine and, to a lesser extent, the N-3 position of cytosine (22). We treated supercoiled plasmid DNA molecules containing Ori with DMS in the presence or absence of Rep and various Rep fragments and mapped modified residues in the Ori sequence by primer extension (Fig. 4).


Figure 4
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  		FIG. 4. DMS footprints of ColE2 Rep and its partial fragments bound to Ori DNA. Supercoiled plasmid DNA carrying Ori was incubated with or without the intact ColE2 Rep protein or its fragments and treated with DMS as described in Materials and Methods. The modified sites on the top and bottom strands were mapped by primer extension, using FITC-labeled primers, on a sequencing gel (left panels). Control sequencing ladders (A, G, T, and C) were obtained by using the same primers. The 37-bp origin region is indicated with an arrow in each gel. Nucleotides protected by Rep against DMS modification are indicated by triangles, and those showing increased DMS sensitivity upon Rep binding are indicated by circles. (A1) No Rep (lane 1), 100 nM Rep (lane 2), and 200 nM Rep (lane 3); (B1) no RepC132 (lane 1), 100 nM RepC132 (lane 2); (C1) no RepC45 (lane 1), 200 nM RepC45 (lane 2), and 400 nM Rep45 (lane 3); (D1) no RepC61 (lane 1), 100 nM RepC61 (lane 2), and 200 nM RepC61 (lane 3); (E1) no RepC94 (lane 1), 100 nM RepC94 (lane 2), and 200 nM RepC94 (lane 3); (F1) no RepC117 (lane 1), 100 nM RepC117 (lane 2), and 200 nM RepC117 (lane 3). Scans of lanes with naked DNA and Rep-bound DNA are shown by gray and black lines, respectively (right panels). Quantification of the experiments shown in the sequencing gels was performed by using NIH Image 1.62 software. A schematization of the 37-bp boxed origin region is presented, and sites I, II, and III bound by the wild-type Rep protein are indicated by the gray background. The symbols are the same as those described for the left panels. (A2) Lanes 1 and 3 of panel A1; (B2) lanes 1 and 2 of panel B1; (C2) lanes 1 and 3 of panel C1; (D2) lanes 1 and 3 of panel D1; (E2) lanes 1 and 3 of panel E1; (F2) lanes 1 and 3 of panel F1.

Binding of the intact ColE2 Rep protein to Ori resulted in the protection of some bases at or close to three sites (I, II, and III) (Fig. 4A). In site I, G at position 6 (6G) on the top strand and 10G and 11G on the bottom strand were protected. In addition, 5T, 7A, and 8G on the top strand and 9T and 12T on the bottom strand seemed to be protected. In site II, 14A, 16A, and 17A on the top strand were significantly protected. In site III, 24A and 25G on the bottom strand were protected. These results indicate that Rep interacts with those bases in sites I and III of Ori from the major groove and with those in site II from the minor groove, based on the specificity of DMS modification. Sites I and II are located roughly adjacent to each other on one surface of dsDNA in the left half of Ori, and site III is located on the opposite surface next to the primer sequence.

Stimulation of DMS modification was detected at 12A and 18G on the top strand and at 19G on the bottom strand. These bases are located at the boundary between sites I and II and just outside site II, suggesting some distortion of dsDNA upon binding of Rep. In site III, modification at 25C on the top strand was greatly stimulated, and modification at neighboring positions (23A, 24T, 26A, 27G, 28A, and 29T) on the top strand was also stimulated. Stimulation at 28A was very weak in the results shown (Fig. 4A). These bases in site III are all on the strand complementary to the template strand for primer synthesis, suggesting possible distortion or opening of dsDNA in site III of Ori upon binding of Rep. Such a conformational change of dsDNA might be required for primer synthesis by Rep. Protection at 6G on the top strand and at 10G, 11G, and 25G on the bottom strand and stimulation at 27G are consistent with the results obtained by using a Rep protein without the His6 tag and linear Ori DNA (H. Matsui and T. Itoh, unpublished data).

Very similar results (Fig. 4B) were obtained with RepC132 (Fig. 1) compared with the intact Rep protein. These results suggested that the C-terminal region of Rep, with 132 residues, is sufficient for interaction with all three sites in Ori and that the N-terminal region is unnecessary for specific Ori DNA binding. RepC132 is also capable of inducing a conformational change of dsDNA at and around site III. RepC37, containing the putative HTH motif alone, was capable of apparently very weak specific binding to Ori, as shown by EMSA (Fig. 2A). In fact, we were able to detect very faint protection in a part of site I in the presence of far excess amounts of RepC37 over Ori in the DMS protection assay (data not shown), suggesting that the interaction between the putative HTH motif alone and Ori was very unstable. On the other hand, when RepC45, containing an additional eight amino acids which are absent in RepC37, was used in the DMS protection assay, the bases in site I were clearly protected (Fig. 4C). The additional amino acids in RepC45 seemed to stabilize the binding of the putative HTH motif to Ori. Based upon the results of EMSA and those of DMS protection assays, we propose that the region from positions 261 to 297 in Rep (region I) contains a DBD, which recognizes and binds to site I of Ori.

To identify the region in Rep involved in binding to site II, we used RepC62, which contains an additional 16 amino acids at the N terminus of RepC45 (Fig. 1). The additional region contains region A of Rep, involved in determining the specificity of Rep-Ori binding in plasmids ColE2-P9 and ColE3-CA38 (29). RepC62 showed clear protection at the bases in site II (Fig. 4D). This suggested that the stable binding of Rep to site II of Ori requires the amino acid residues at positions 236 to 252. We propose that the second binding domain is located to the right of position 236 and down to around position 260 in Rep (region II), making a contact with the Ori DNA from the minor groove. A closer look at the results with RepC62 revealed weak but significant stimulation at 24A and 25G in site III on the bottom strand by binding of RepC62 (Fig. 4D). This suggested that the binding of RepC62 to sites I and II induced destabilization of dsDNA in site III, approximately one turn apart.

The results presented above also allowed us to narrow the region in Rep containing the DNA binding domain interacting with site III of Ori, which is to the right of position 166 (tentative region III). To further delimit the region containing the third domain, we used two additional deletion Rep proteins with deletion end points to the right of position 166, namely, RepC94 and RepC117 (Fig. 1). The result with RepC94 was essentially similar to that with RepC62, except that an additional residue (20G) on the bottom strand between sites II and III was protected (Fig. 4E). This might result from interaction of the additional amino acids in RepC94 with the Ori DNA. RepC117 was capable of binding to site III, in addition to sites I and II, and also of inducing a conformational change of dsDNA in and around site III (Fig. 4F), although the interaction with site III was weaker than those with RepC132 and the wild-type Rep protein. This suggested that the region binding to site III of Ori is present to the right of position 180 and that the presence of the region from positions 166 to 180 stabilizes the interaction of Rep with site III of Ori. A notable amino acid sequence, GLGRN, is present in this region (Fig. 1C), in which the first G and the sequence GRN are strictly conserved in the Rep proteins of the ColE2-related plasmids (11). We constructed mutant Rep proteins with amino acid substitutions at these positions (G172E, G174W, R175Q, and N176D) by random mutagenesis using Mn2+ PCR. All of these mutant Rep proteins were defective in initiating in vivo plasmid ColE2 DNA replication (data not shown), indicating the importance of the conserved residues (G-GRN) for the activity of Rep. The binding affinity of RepN176D for the Ori DNA was apparently similar to that of the intact Rep protein, as examined by EMSA (Fig. 5A). The result of a DMS protection assay using RepN176D (Fig. 5B) was similar to that using wild-type Rep or RepC132 (Fig. 4A1 and 4B1), except that the protection of the bases on the bottom strand at site III by RepN176D was hardly detectable. Note that the modification of bases on the bottom strand at site III of Ori was stimulated when site III was not bound by Rep, as observed with RepC94 and RepC62 (Fig. 4D and E). RepN176D was also fully capable of inducing a conformational change of dsDNA at and around site III, as revealed by stimulation of modification of bases by DMS on the top strand at site III. These results supported the notion that the presence of the region (positions 166 to 180) containing position 176 stabilizes the interaction of Rep with site III of Ori. Weaker protection on the bottom strand and stronger stimulation of some bases on the top strand at site III by RepN176D than by RepC117 (compare Fig. 5B and Fig. 4F1) might result from the presence of some region in the remaining N-terminal half of Rep that is absent from RepC117 which further induces a conformational change of dsDNA at and around site III of Ori.


Figure 5
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  		FIG. 5. Specific Rep-Ori DNA binding of mutant Rep protein and mutant origins. (A) EMSA using the wild type and RepN176D was performed as described in the legend to Fig. 3. (B) DMS protection assays, with (+; 100 nM) or without (–) RepN176D, were performed as described in the legend to Fig. 4. (C and D) DMS protection assays using supercoiled plasmid DNAs carrying Oridr56 (C) and Oridr4 (D), with (+) or without (–) RepC132, performed as described above.

Potential linker region connecting the binding domains of Rep. In order to analyze the structures and functions of various regions of Rep, we constructed mutant Rep proteins with single amino acid substitutions all through, from the N terminus to the C terminus, by random mutagenesis and tested their replication activities (the details will be described elsewhere). We obtained many mutant Rep proteins defective in replication activity whose mutations were located in the regions important for origin binding (Fig. 1C). These amino acids seem to be involved directly in the origin binding activity of Rep or in formation of the proper structures of these regions. On the other hand, we noticed a region (around positions 191 to 211) for which almost all of the mutant Rep proteins obtained (14 mutants with substitutions at 13 positions) retained full replication activity, although in some of them the properties of amino acids were significantly changed by the mutations (Fig. 1C). These results suggested that the original amino acid sequence in this region is not important for Rep activity. We propose that this region might function as a linker connecting the two regions (domains) of Rep involved in Ori binding. Furthermore, RepC94, with a deletion end point in the putative linker region, protected the residue (20G) on the bottom strand between sites II and III (Fig. 4E), suggesting the possibility that the putative linker region crosses over the major groove between sites II and III. The presence of a putative linker region (around positions 190 to 211) further delimits region III, containing the third DBD, between positions 166 and 190.

Interaction of region III of Rep with site III of Ori. To further analyze the interaction of region III of Rep with site III of Ori, we used a deletion mutant origin (Oridr8) (Fig. 1A and Table 2) lacking site III in EMSA (Fig. 6A). RepC132 bound to Oridr8 much less efficiently than it bound to the intact origin (Fig. 6A, lanes 2 and 3). RepC117, however, bound to Oridr8 and the intact origin with roughly similar affinities, which might be due to unstable binding of RepC117 to site III of Ori. The DMS protection assay revealed that RepC117 interacted with Oridr8 much less efficiently than it interacted with the intact origin (data not shown). In contrast to the protection signals at three sites of the intact origin (Fig. 4F), only site I of Oridr8 was protected weakly. Both RepC94 and RepC62 bound to the intact origin and Oridr8 with similar affinities (Fig. 6A). Note that they protected only sites I and II of Ori in DMS protection assays (Fig. 4D and E). These results suggested the possibility that region III of Rep forms an independent functional binding domain and interacts with site III of Ori. The results with RepC94 and RepC62 suggested that when region III of Rep is missing, the interactions of the deletion Rep proteins with sites I and II of Ori are not affected by the presence or absence of site III. The additional amino acid sequence in RepC117 might interfere with the interactions of regions I and II of Rep with sites I and II of Ori when site III of Ori is missing.


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  		TABLE 2. Mutant origins from the right


Figure 6
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  		FIG. 6. Specific binding of various Rep fragments to the wild-type Ori and Oridr8, as analyzed by EMSA. (A) Various Rep fragments were incubated with end-labeled wild-type Ori and Oridr8 fragments. The concentration of each Rep fragment was adjusted to the level that was unable to saturate the origin DNA. The protein-DNA complexes (indicated by circles) and free DNA fragments were analyzed as described in the legend to Fig. 3. (B) Schematic representations of various Rep fragments bound to the wild-type Ori and Oridr8. Sites I, II, and III of Ori are indicated below the boxes representing the binding sites of the origin region (shown by lines). Regions I, II, and III of Rep are indicated in the ovals representing the domains of Rep.

Sequence to the right of site III of the minimal region of Ori. The deletion mutants of Ori lacking the sequence to the right of site III (Oridr56 and Oridr4) failed to initiate DNA replication (35). Nevertheless, they retained similar and slightly weaker IncB activities than the intact origin, suggesting that these two deletion origins are capable of binding to Rep as efficiently as the intact origin. We do not know, however, whether they interact with Rep in the same mode as that of the intact origin, and some difference in the binding mode could affect the initiation of DNA replication at the origin. To address this problem, we used RepC132, having a similar binding activity to that of the intact Rep protein (Fig. 4A and B) but showing more solubility (data not shown), and supercoiled plasmid DNAs containing Oridr56 and Oridr4 in DMS protection assays (Fig. 5C and D). Both of the origins gave similar results to that for the intact origin DNA (Fig. 4B1). Namely, three sites (I, II, and III) were protected, and a conformational change of dsDNA at and around site III was detected. These results suggested that the sequence to the right of site III of Ori does not affect the binding mode of Rep and Ori, and we propose that the sequence to the right of site III is essential for the initiation of replication after the step of binding of Rep to Ori. Nucleotides 26T and 27G on the bottom strand next to site III of Oridr4 were also strongly modified by DMS, due to the presence of G instead of C at position 27. This guanine residue, along with the neighboring thymine residue, was protected by the binding of RepC132, supporting the notion that Rep interacts with site III of the origin DNA from the major groove.


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DISCUSSION
 
We identified three regions in the C-terminal region of Rep important for the Rep-Ori interaction, with two of them (regions I and II) interacting with the origin DNA from the adjacent major (site I) and minor (site II) grooves in the left half of Ori and the remaining region (III) interacting with site III, next to the template sequence for primer synthesis from the major groove, which is one and one-half turn apart from site I on the opposite surface of the origin DNA (Fig. 7A and B). We propose that the interaction of region III of Rep with site III of Ori positions the primase domain in the N-terminal region of Rep properly on the primer template. Sites I, II, and III, which were identified by the DMS protection assay, are included within the three Rep protein binding elements (sites a, b, and c, respectively) proposed by in vivo analyses of mutant origins with deletions and single-base-pair substitutions (35). We also identified, by mutation analysis, a potential linker region between regions II and III of Rep which is proposed to cross over the major groove between sites II and III of Ori. Region I of Rep, containing a potential HTH motif based on amino acid sequence homology to known E. coli DNA binding proteins, is located at the C terminus of Rep. We showed here that the region is essential for the specific binding of Rep to Ori. How regions II and III of Rep bind to Ori is still unclear.


Figure 7
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  		FIG. 7. Summary and schematic representation of the results of the DMS protection assays shown in Fig. 5 (A) and a model of Rep-Ori interaction (B). Rep binding sites I, II, and III of Ori are shown above a B-form DNA double helix structure, and regions I, II, and III of Rep interacting with sites I, II, and III of Ori are indicated by rectangles.

We found that the ColE2 Rep protein has sequence homology and secondary structure similarity with the {gamma}{delta} resolvase (36), based on alignment analysis (Fig. 8A). Only the C-terminal regions of these proteins are shown here. Although the entire sequence homology was very low, the binding regions I and II of the ColE2 Rep protein showed homology with the residues within the HTH motif and the extended arm region of the {gamma}{delta} resolvase important for the binding to site I of the res DNA. The crystal structure of the {gamma}{delta} resolvase dimer in complex with the 34-bp site I DNA has been determined (36). Each {gamma}{delta} resolvase monomer has an N-terminal catalytic domain (positions 1 to 120), a three-helix-bundle C-terminal DNA binding domain (positions 148 to 183) that binds specifically to the recognition sequence from the major groove, and an extended arm region (positions 121 to 147) that connects the two domains and interacts with DNA from the adjacent minor groove. The arm region contains an extended turn and a helix. It is worth mentioning that the left half of res DNA site I has sequence similarity to the left half of the plasmid ColE2 origin (Fig. 8B). An intriguing possibility is that the C-terminal DNA binding domains of the ColE2 Rep protein (region I, containing a putative HTH motif, and region II, containing a turn and an {alpha} helix, based on secondary structure prediction) might adopt DNA binding modes related to those of the monomer of the {gamma}{delta} resolvase (a three-helix-bundle DBD and an extended arm region). A GRK sequence in the turn region (extended turn region) of the {gamma}{delta} resolvase is also found in the putative turn region of ColE2 Rep (Fig. 8A). G and R are directly involved in binding to res DNA site I from the minor groove. Similarly, the GRK sequence in ColE2 Rep might interact with the ColE2 origin DNA from the minor groove. A closer look at the results of the DMS protection assay with RepC45, which contains the GRK sequence at the N-terminal end, revealed that 16A in site II appeared to be protected, though very weakly (Fig. 4C).


Figure 8
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  		FIG. 8. Comparisons of ColE2 Rep protein and {gamma}{delta} resolvase and of their cognate binding sites. (A) Alignment of amino acid sequences of ColE2 Rep and {gamma}{delta} resolvase. Identical residues between the two sequences are indicated. The predicted and known secondary structures are shown above and below the amino acid sequences of the ColE2 Rep protein and the {gamma}{delta} resolvase, respectively. H, {alpha} helix; E, ß strand; C, coil. The three-helix-bundle DNA binding domain (containing an HTH motif) and the extended arm region are marked with double-headed arrows below the secondary structure of the {gamma}{delta} resolvase, and helix E and the HTH motif are shaded. The three regions (I, II, and III) of the ColE2 Rep protein important for the origin DNA binding deduced from DMS footprint analysis and the putative linker region deduced from a genetic analysis are indicated above the secondary structure of the ColE2 Rep protein with double-headed arrows and a bracket, respectively, and the putative HTH motif is shaded. Among the residues in the HTH motif of the {gamma}{delta} resolvase involved in binding to site I of the res DNA, those involved directly in base-specific interaction from the major and minor grooves are indicated by outlined letters, while the other residues involved are underlined. (B) Alignment of the nucleotide sequences of the ColE2 origin DNA and site I of the res DNA bound by {gamma}{delta} resolvase. The regions with high homology are highlighted in gray, and the identical residues are indicated by vertical lines. The primer sequence in the ColE2 origin is indicated by the arrow above the sequence. Twelve-base-pair inverted repeats in site I of the res DNA are indicated by arrows below the sequence. The regions recognized and bound from the major and minor grooves by the C-terminal binding domains of the {gamma}{delta} resolvase are shown by brackets below the sequence, while Rep binding sites I and II of the ColE2 origin identified in this study are shown by brackets above the sequence.

The presence of the region from positions 166 to 180 within region III stabilizes the interaction of Rep with site III of Ori. Interestingly, the GLGRN sequence in this region, which is important for Rep activity, as shown above, and which is conserved in the Rep proteins of the ColE2-related plasmids (11), shows homology to the RLGRD sequence in the catalytic domain of {gamma}{delta} resolvase (Fig. 8A). The second arginine in the RLGRD sequence of the monomer of {gamma}{delta} resolvase is involved in catalysis and is directly hydrogen bonded to the DNA backbone (36). Similarly, the arginine in the GLGRN sequence of ColE2 Rep might interact with the DNA backbone to stabilize the Rep-Ori binding.

RepC117, lacking the region from positions 166 to 180, bound to the three sites of Ori like the intact Rep protein did, although the protection of site III of Ori by RepC117 was weaker than that by the intact Rep protein in DMS protection assays (Fig. 4A and F). The origin-melting activity by RepC117 was very low compared with that by the intact Rep protein (M. Han, K. Aoki, M. Yagura, and T. Itoh, unpublished data). This raised the possibility that the region (positions 166 to 180) is important for the in vivo replication of plasmid ColE2, not merely in stabilizing the interaction of Rep with dsDNA of Ori as a part of the third binding domain. We speculate that this region might stabilize the open complex structure by binding to single-stranded DNA of the melted region, which is important for progress of primer synthesis by Rep.

The effective lengths of binding regions on DNA where DNA binding proteins interact depend upon the structures, sizes, and numbers of the DNA binding domains and also upon whether the proteins interact as monomers, dimers, or oligomers (6). In most plasmid replicons, such as the iteron-regulated plasmids (F, pSC101, pPS10, miniP1, etc.), the Rep proteins consist of about 300 amino acids, and the Rep binding sites in the origins are about 20 bp long (2, 5). The Rep54 monomer of plasmid F has extensive polar interactions between the two recognition helices of two DNA binding domains and two adjacent major grooves on one surface of the iteron DNA (19). The Rep monomers of pPS10, pSC101, and miniP1 were suggested to have similar structures and might bind to their iterons similarly (28). The RepA monomer of pPS10 was suggested to have an extended form suitable for iteron binding by biochemical analysis (4, 7). The Rep binding site in the ColE2 origin is about 20 bp long, based on the DMS protection assay, which is relatively long for the 132-amino-acid C-terminal region of ColE2 Rep, if we assume a compact globular form. Therefore, a rather extended conformation of the C-terminal region of ColE2 Rep appears to be likely. We recently found that Rep binds to Ori as a monomer (Han et al., unpublished data). The nucleotide sequences of sites I, II, and III in the ColE2 origin are asymmetric. This would require an asymmetric conformation of the DBDs of Rep, and a monomer with three separate DBDs would be the simplest form of Rep suitable for this requirement.

Linker regions covalently connecting two DNA binding domains in single proteins are found in the prokaryotic transcriptional activators of the AraC family. In MarA, which belongs to the AraC family, two helix domains are linked by an {alpha}-helix that appears to provide the flexibility necessary for the two domains to interact with two successive major grooves of the DNA (25). Such linker regions in DNA binding proteins are more commonly found in eukaryotic systems. In mammalian transcription factors, such as Oct-1 and POU homeodomain proteins, for example, two DBDs are connected by a linker region, which serves as a flexible tether between the two domains and permits different relative arrangements of the two domains suitable for the binding sites on the DNA (27). The potential linker region of ColE2 Rep is predicted to form an {alpha} helix (Fig. 1B) and has sequence homology with helix 2 of the Iß subdomain of the MuA end binding domain (Iß{gamma}) (Fig. 9). The Iß subdomain can be divided into two structural elements, and helix 2 serves to bridge these two elements (26). The putative linker region of ColE2 Rep might form a similar structure, linking two DNA binding domains on both sides and allowing Rep to bind to Ori properly.


Figure 9
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  		FIG. 9. Alignment of the putative linker region of the ColE2 Rep protein and helix 2 of the Iß subdomain of the MuA transposase. The putative linker region of the ColE2 Rep protein deduced from the mutation analyses in this paper is indicated by a bracket above the sequence. The predicted {alpha} helix region of the ColE2 Rep protein and the known {alpha} helix region (helix 2) of the Iß subdomain are indicated by solid bars.

The ColE2 Rep protein is a multifunctional initiator, although it is a small protein. The N-terminal half of Rep is unnecessary for origin DNA recognition, as shown in this paper, whereas it is indispensable for replication of the plasmid ColE2. Rep is a primase which can synthesize a three-nucleotide primer RNA specific for the ColE2 origin, which is utilized by the host DNA polymerase I to initiate replication. Therefore, the N-terminal region must contain the primase domain. We are interested in how Rep binds to Ori to perform its multiple functions. The findings in this paper should help us to understand the early steps of initiation of plasmid ColE2 replication in more detail in future analyses.


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ACKNOWLEDGMENTS
 
We are grateful to all of the members of our research group for their discussions. We also thank K. Aoki for providing the KA1 primer and S. Nishio for the figure of the DNA double helix structure. We thank G. Minote and N. Hayashida of the Research Center for Human and Environmental Sciences of Shinshu University for radioisotope experiments and for providing us an opportunity to use a Shimadzu DNA sequencer.

This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano 390-8621, Japan. Phone: 81-263-37-2489. Fax: 81-263-37-2560. E-mail: tateito{at}gipac.shinshu-u.ac.jp. Back

{triangledown} Published ahead of print on 10 November 2006. Back


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Journal of Bacteriology, February 2007, p. 1061-1071, Vol. 189, No. 3
0021-9193/07/$08.00+0     doi:10.1128/JB.01455-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Aoki, K., Shinohara, M., Itoh, T. (2007). Distinct Functions of the Two Specificity Determinants in Replication Initiation of Plasmids ColE2-P9 and ColE3-CA38. J. Bacteriol. 189: 2392-2400 [Abstract] [Full Text]  

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