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Journal of Bacteriology, June 2004, p. 3785-3793, Vol. 186, No. 12
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.12.3785-3793.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Role of RepA and DnaA Proteins in the Opening of the Origin of DNA Replication of an IncB Plasmid

T. Betteridge, J. Yang, A. J. Pittard, and J. Praszkier*

Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia

Received 8 December 2003/ Accepted 1 March 2004


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ABSTRACT
 
The replication initiator protein RepA of the IncB plasmid pMU720 was shown to induce localized unwinding of its cognate origin of replication in vitro. DnaA, the initiator protein of Escherichia coli, was unable to induce localized unwinding of this origin of replication on its own but enhanced the opening generated by RepA. The opened region lies immediately downstream of the last of the three binding sites for RepA (RepA boxes) and covers one turn of DNA helix. A 6-mer sequence, 5'-TCTTAA-3', which lies within the opened region, was essential for the localized unwinding of the origin in vitro and origin activity in vivo. In addition, efficient unwinding of the origin of replication of pMU720 in vitro required the native positioning of the binding sites for the initiator proteins. Interestingly, binding of RepA to RepA box 1, which is essential for origin activity, was not required for the localized opening of the origin in vitro.


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INTRODUCTION
 
Miniplasmid pMU720, a derivative of a large, low-copy-number, conjugative plasmid, pMU707, belongs to incompatibility group B (3). The frequency of replication initiation of pMU720 depends on the expression of repA, the gene for plasmid initiator protein RepA. The RepA protein, whose synthesis is controlled at the translational level by a small 71-base antisense RNA, RNAI, is essential and rate limiting for replication of pMU720 (39, 48, 49, 56-58).

Although in vivo RepA acts preferentially in cis, it is able to activate its ori in trans: i.e., when it is present on a second plasmid (40). However, replication from an ori present in trans appears to be relatively inefficient, requiring significantly higher levels of RepA than activation of ori in cis (40). Activation of ori in trans did not require the presence of CIS, the sequence separating the repA coding sequence and ori in pMU720 and thought to be involved in tethering the nascent RepA protein and loading it onto the ori in cis (40, 41).

RepA has been purified and used in vitro to elucidate its binding sites in ori (2). The protein was found to bind to a region of ori containing four copies of the sequence motif 5'-AANCNGCAA-3'. Mutagenic analyses demonstrated that this sequence represents the binding site of RepA (the RepA box) and that only three of those sites, RepA boxes 1, 2, and 4, are essential for origin activity in vivo. RepA protein binds to the RepA boxes in an ordered and sequential manner, with RepA box 1 occupied first and RepA boxes 3 and 4 occupied last. The spacing between RepA boxes is also critical for origin activity in vivo, suggesting that RepA molecules bound to ori may interact with one another and that this interaction may be required for optimal origin function (2).

The ori of pMU720 contains the sequence 5'-TTATCCACA-3', which matches the stringent consensus sequence for a DnaA binding site, the DnaA R box. This sequence is required for efficient origin activity in vivo, as its deletion resulted in an ~3-fold reduction in copy number, both when RepA was provided in cis and when it was provided in trans (40, 41). DnaA is the initiator protein of chromosomal DNA replication in Escherichia coli and other eubacteria. DnaA binds to the five DnaA R boxes (R1 to R4 and M) in the origin of replication of E. coli (oriC) in an ordered and sequential manner, introducing a 40° bend at each binding site (29, 45, 54). In addition, DnaA binds to three other 9-mers in oriC, the I sites, which deviate from the consensus sequence of the R box by 3 to 4 nucleotides (nt) (13, 42). The accumulated topological stress unwinds the oriC at the AT-rich region present to the left of DnaA box R1, to form an open complex. The ATP-DnaA form of DnaA then binds to the single-stranded 6-mer sequences 5'-AGATCT-3' in the unwound region, stabilizing the single-stranded region in preparation for loading of the DnaB helicase (50, 51). Helicase, in the form of DnaB-DnaC complex, is recruited to the unwound region of oriC by DnaA, which makes direct contact with DnaB in the complex (30, 31, 55).

Unlike chromosomal replication, which requires a single initiator protein, DnaA, many bacterial plasmids require combined actions of DnaA and a plasmid-encoded initiator protein for their replication. For most of these plasmids, the requirement for DnaA is absolute, as in the case of plasmids pSC101, F, Rts1, the {gamma} ori of R6K, RK2, and mini-P1, which cannot replicate in E. coli in the absence of DnaA (11, 14, 15, 17, 21, 28). However, in some plasmids, such as the IncFII plasmids NR1 and R1, DnaA appears to play only an auxiliary role, as these plasmids can replicate in a dnaA-null host, albeit with reduced efficiency (35, 52). All plasmids showing absolute or partial dependence on DnaA contain one or more copies of the DnaA R box in their origin of replication. Analyses of the role of DnaA in replication of these plasmids revealed that it is different for different plasmids but involves participation in open complex formation, recruitment of DnaB helicase, or both (8, 19, 23, 24, 27, 37, 38, 46).

pMU720, unlike other theta-replicating plasmids of gram-negative bacteria that have been characterized to date, does not belong to the family of iteron-regulated plasmids, but is a member of the extended family of IncFII-related replicons whose copy number is regulated by an antisense RNA. The two families of plasmids differ in many other aspects of their basic biology. Thus, the Rep proteins of the iteron-regulated plasmids are trans-acting and exist in solution as dimers, which must be converted to monomers, the replication-proficient form of the protein. In contrast, the Rep proteins of the IncFII-related replicons are cis-acting and there is no evidence for the existence of two forms of the protein, only one of which is active in replication. In the IncFII-related replicons, synthesis of Rep proteins is rate limiting for replication so that an increase in expression of rep results in an increase in copy number, whereas there is no such direct correlation between expression of rep and copy number in the iteron-regulated plasmids, and in many cases it has been shown that increased synthesis of Rep has an inhibitory effect on replication. The binding sites for Rep in ori of the iteron-regulated plasmids are ~20 bp long, whereas those of the IncFII-related replicons are ~9 bp long (2, 9). In view of these characteristics of the Rep proteins of the two plasmid families, it is perhaps not surprising that amino acid homology and/or protein structure comparisons have shown that the initiators of most of the well-characterized iteron-regulated plasmids are related to RepE of plasmid F (22, 47), whereas those of the IncFII-related replicons are related to RepA of plasmid R1 (36).

In the iteron-regulated plasmids, the site in ori for open complex formation is an AT-rich region, usually containing repeated sequences, lying in proximity to the region that contains binding sites for Rep. However, no such region was apparent upon examination of the sequence of ori of pMU720 (oriB), and there have been no previous studies of open complex formation in IncFII-related replicons. This paper represents the first such study, describing the analysis of requirements for open complex formation in the ori of pMU720. It was found that binding of the RepA protein of pMU720 to its cognate ori resulted in open complex formation at a specific sequence lying immediately downstream of RepA box 4. Although DnaA was able to bind to the DnaA R box in oriB independently of RepA, it could not induce unwinding at this origin. However, DnaA enhanced the localized unwinding of oriB by RepA. Efficient unwinding of oriB in vitro required the presence of both RepA and DnaA, the native positioning of the RepA boxes, and the presence of the 6-mer 5'-TCTTAA-3' downstream of RepA box 4.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and phages. The strains of E. coli K-12 used in this study are given below. JM101 [{Delta}(lac-proAB) supE thi F' (traD36 proA+B+ lacIqZ{Delta}M15)] (33) was used for cloning and propagating M13 derivatives. XL1 Blue MRF' {Delta}(mcrA)183 {Delta}(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F' proAB lacIqZ{Delta}M15 Tn10 (Tetr)] (Stratagene) was used to grow M13 derivatives that had undergone mutagenesis as described by Vandeyar et al. (53). JP3438 (thr-1 leuB6 thi-1 lacY1 gal-351 supE44 tonA21 hsdR4 rpoB364 recA56) was used for propagating pMU720 derivatives and for all copy number determinations.

Bacteriophage vectors used to clone fragments for DNA sequencing and mutagenesis were M13tg130 and M13tg131 (20). The plasmids used are described in Table 1.


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  		TABLE 1. Plasmids used in this study

Media, enzymes, and chemicals. The minimal medium used was half-strength buffer 56 (34) supplemented with 0.2% glucose, thiamine (10 µg/ml), and necessary growth factors. Enzymes and chemicals of a suitable grade were purchased commercially and not purified further. [{alpha}-35S]dATP (>1,000 Ci/mmol) for use in sequencing and [{gamma}-32P]ATP (5,000 Ci/mmol) for end-labeling DNA fragments were obtained from Amersham Biosciences Pty., Ltd. Ampicillin was used at a final concentration of 50 µg/ml, chloramphenicol was used at 10 µg/ml, kanamycin was used at 20 µg/ml, isopropyl-ß-D-thiogalactopyranoside (IPTG) was used at 1 mM, and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) was used at 25 µg/ml.

Recombinant DNA techniques. Plasmid and bacteriophage DNA were isolated and manipulated as described by Sambrook and Russell (43). DNA was sequenced with a model 377 DNA sequencer and ABI Big Dye terminators (Perkin-Elmer Corporation) or by the method of Sanger et al. (44), modified in that T7 DNA polymerase was used instead of the Klenow fragment and terminated chains were uniformly labeled with [{alpha}-35S]dATP. Oligonucleotide-directed in vitro mutagenesis reactions were performed on single-stranded M13 templates, using the method of Vandeyar et al. (53). Oligonucleotides were purchased from GeneWorks, Ltd. DNA sequencing was used to screen for and confirm the presence of mutations.

Purification of the RepA and DnaA proteins. Recombinant RepA protein was overexpressed and purified as described previously (2). Recombinant DnaA-His6 protein was overexpressed and purified as described previously (6), except that 20 mM imidazole was included in sonication and wash buffers. Purified DnaA was activated before use by incubation on ice for 15 to 30 min with 5 mM ATP in HEPES buffer [25 mM HEPES-KOH (pH 7.6), 10 mM Mg(OAc)2, 4 mM dithiothreitol (DTT), 1 mM EDTA, 0.2% Triton X-100, and 0.6 mg of nuclease-free bovine serum albumin per ml].

Labeling of primers. Primers TN96 (5'-CCAGTGAATTGCTGCAGAGATC-3'), TN97 (5'-GTTCACAGTGGTTTCAGAGAT-3'), and TN98 (CCACGCATCAGTCATCAGAACGTGG-3'), were labeled at the 5' end with [{gamma}-32P]ATP and T4 polynucleotide kinase (Promega).

DNase I footprinting experiments. The DNA fragments for DNase I footprinting were amplified by PCR using M13 derivatives carrying the wild-type or mutant ori region of pMU720 and 10 pmol of primers TN96 and TN97; primer TN97 was labeled for analysis of the bottom strand and TN96 was labeled for analysis of the top strand. The 190-bp labeled fragment was purified on a native polyacrylamide gel. The end-labeled fragment was mixed with 90 to 720 nM RepA protein and/or 2 to 60 nM DnaA protein in a binding buffer consisting of 50 mM Tris-HCl (pH 7.8), 50 mM NaCl, 3 mM Mg(OAc)2, 0.1 mM DTT, 0.1 mM EDTA, 0.1 mM CaCl2, 1 mM ATP, 0.2 µg of poly(dI-dC), and 0.6 mg of bovine serum albumin per ml; equilibrated at room temperature for 10 min, and then incubated at 30°C for 20 min. A standard binding reaction mixture contained 2 to 10 ng of labeled DNA (20 to 160 fmol) in a total volume of 25 µl. The protein-DNA complexes were digested with 0.0125 U of DNase I (Amersham Biosciences Pty., Ltd.) for 30 s at room temperature, and 10 µl of phenol and 15 µl of chloroform were added to stop the reaction. Samples were extracted and ethanol precipitated. The pellets were resuspended in formamide dye mix and analyzed on a 6% polyacrylamide sequencing gel. Following electrophoresis, the gel was scanned with a phosphorimager (Fuji FLA-3000G) and then exposed to Kodak XAR film at –70°C for 48 h.

KMnO4 footprinting. Supercoiled plasmid templates carrying oriB, purified with a Qiagen plasmid Maxi kit (Qiagen Tip-500), were mixed with 220 or 440 nM RepA protein and/or 40 nM DnaA protein in binding buffer consisting of 50 mM Tris-HCl (pH 7.8), 50 mM NaCl, 3 mM Mg(OAc)2, 0.1 mM DTT, 0.1 mM EDTA, and 0.6 mg of bovine serum albumin per ml. A standard binding reaction mixture contained 1 µg of supercoiled DNA in a total volume of 40 µl. When it was added, the final concentration of ATP was 1 mM. Preincubation for 20 min at room temperature with RepA was carried out before the addition of DnaA. The reaction then was shifted to 37°C for another 20 min. KMnO4 was added to final concentration of 6 mM. After a further 2-min incubation at 37°C, the reaction was stopped by the addition of 3 µl of 98% ß-mercaptoethanol and EDTA to a final concentration of 40 mM. Samples were purified by phenol-chloroform extraction and ethanol precipitation and then linearized with restriction endonuclease NheI, phenol-chloroform extracted, and ethanol precipitated again. An aliquot was analyzed on a 1% agarose gel to quantify the DNA.

Primer extension reactions were carried out in a Thermal Cycler for 25 cycles, using PCR Mastermix (Promega Corporation) and appropriate {gamma}-32P-labeled primer. Primer TN97 was used to detect KMnO4-modified residues on the top strand, and primer TN98 was used to detect the modified residues on the bottom strand. Approximately 0.75 pmol (0.5 ng) of labeled primer and 50 ng of KMnO4-modified template were used per reaction.

Construction of plasmids for use in copy number determination. The two-plasmid system (40) was used to study in vivo the interactions of RepA with oriB in trans. The RepA-producing plasmid was pMU1585 (40), and the ori plasmid was pMU1600 (2) or its derivatives carrying mutations in the oriB sequence. Plasmid pMU1600 contains the modified pMB1 replicon from pAM34 (12), in which the essential preprimer RNA is transcribed from the lacZ promoter. Since this plasmid contains the lacIq gene, replication of its pAM34 replicon requires the presence of a lac inducer, such as IPTG (isopropyl-ß-D-thiogalactopyranoside). Thus, in the absence of IPTG, replication of the ori plasmids is dependent on ability of oriB to be activated by the RepA protein provided in trans by the RepA-producing plasmid. Failure of cells cotransformed with the ori and RepA-producing plasmids to grow in the absence of IPTG was scored as the inability of the ori to support plasmid replication. The presence of a constitutively expressed chloramphenicol acetyltransferase (CAT) reporter gene allows estimations of the copy numbers of the ori plasmid and its derivatives to be made.

Introduction of ori and RepA plasmids into E. coli cells. ori and RepA plasmids were cotransformed into E. coli K-12 strain JP3438 by the method of Chung et al. (7). Cells were plated onto medium containing half-strength buffer 56 (34), 0.2% glucose, 0.2% Casamino Acids, thiamine (10 µg/ml), ampicillin, chloramphenicol, and kanamycin, with and without IPTG, and incubated for 72 h at 37°C. Plates were checked after 48 and 72 h of incubation, and the number and size of colonies produced in the presence and absence of IPTG were compared.


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RESULTS
 
Characterization of binding of DnaA protein to the origin of replication of pMU720. The presence of a DnaA R box in oriB suggests that this host protein may have a role in initiation of replication of the IncB plasmid. DNase I footprinting was used to characterize the binding of DnaA protein to the bottom strand of oriB. It was found that DnaA was able to bind to oriB independently of RepA, protecting an 18-bp region lying between nt 1912 and 1929 (Fig. 1A, lane 1), which encompasses the DnaA R box (nt 1920 to 1928), and promoting the appearance of a weak hypersensitive band at position 1926. Increasing the DnaA concentration fourfold did not alter the footprint (data not shown), indicating that at 15 nM DnaA was already present at an optimal level. DNase I footprinting was used to determine whether the presence of DnaA affected binding of RepA to oriB. It was found that whereas in the absence of DnaA, protection of all three RepA boxes (nt 1926 to 1993) and the appearance of hypersensitive bands at positions 1949 and 1950 required 720 nM Rep protein (Fig. 1A, lane 4), in the presence of DnaA, a comparable level of protection was already seen with 280 nM RepA protein (Fig. 1A, lane 6), and increasing the concentration of RepA to 720 nM resulted in significantly stronger protection (Fig. 1A, lane 7). The footprint produced by the two proteins was a summation of their separate footprints, except that the presence of RepA in the reaction resulted in the enhancement of the hypersensitive site at position 1926, within the DnaA R box (Fig. 1A, lanes 5 to 7).



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  		FIG. 1. Analysis of RepA and DnaA interactions with wild-type oriB. Double-stranded oriB fragment, 5'-end labeled in the bottom strand, was incubated with RepA and/or DnaA protein and subjected to partial digestion with DNase I. Regions of DNA protected from digestion by DNase I are indicated by vertical lines, with the positions of the first and last bands protected shown. Asterisks indicate sites of enhanced cleavage by DNase I. DB, DnaA R box; RB1 to RB4, Rep A boxes; A+G, Maxam-Gilbert sequencing reaction. (A) Lanes: control, no RepA or DnaA; 1, no RepA; 2 and 5, 90 nM RepA; 3 and 6, 280 nM RepA; 4 and 7, 720 nM RepA. DnaA was present in lanes 1, 5, 6, and 7 at 15 nM. (B) Lanes: control, no RepA or DnaA; 1 and 6, 2 nM DnaA; 2 and 7, 4 nM DnaA; 3 and 8, 8 nM DnaA; 4 and 9, 16 nM DnaA; 5 and 10, 32 nM DnaA. RepA was present in lanes 6 to 10 at 720 nM.

To determine whether RepA affected binding of DnaA to oriB, the ability of suboptimal concentrations of DnaA to protect the oriB fragment from digestion by DNase I in the presence and absence of RepA was examined. It was found that whereas in the absence of RepA, complete protection of the DnaA R box (nt 1920 to 1928) and the appearance of a hypersensitive band at position 1926 required 16 nM DnaA protein (Fig. 1B, lane 4), in the presence of RepA, complete protection was already seen with 2 nM DnaA protein (Fig. 1B, lane 6), the lowest concentration of DnaA tested. Thus, the two initiator proteins show reciprocity, each enhancing the binding of the other to oriB.

Role of RepA and DnaA in localized unwinding of the origin of replication of pMU720. Binding of the initiator proteins to their specific recognition sequences in the origin of replication leads to structural distortion of the DNA and results in localized unwinding of the DNA. Since both DnaA and RepA bound to their specific recognition sequences in oriB, the contributions of each of these initiator proteins to the localized unwinding of the oriB DNA were assessed by using KMnO4 footprinting. KMnO4 reacts preferentially with pyrimidine residues in single-stranded DNA, oxidizing primarily T residues. The modified residues in supercoiled DNA can be detected by primer extension, because chain elongation terminates at these residues. Figure 2 shows the primer extension products from the top and bottom strands of wild-type oriB template after incubation with RepA and/or DnaA and subsequent KMnO4 treatment. There was no detectable modification of the supercoiled oriB DNA by KMnO4 in the absence of the initiator proteins (Fig. 2A and B, lane 1) nor when RepA and DnaA were present but KMnO4 was absent (Fig. 2A and 2B, lane 2). Absence of detectable KMnO4 modification following binding of DnaA showed that, alone, this protein was unable to induce unwinding of oriB (Fig. 2A and B, lane 4). In contrast, RepA induced localized unwinding of oriB, with T1993 and T1994 in the top strand and T1987, T1989, and T1995 in the bottom strand (Fig. 2A and B, lane 3) oxidized by KMnO4. In addition, termination was detected at residues T1942 and T1943 on the bottom strand (Fig. 2B, lane 3) but no equivalent modification was seen on the top strand. Thus, there are two regions in oriB that become sensitive to KMnO4 modification upon binding of RepA: one positioned in the spacer between RepA boxes 1 and 2 (region I, Fig. 3) and the other positioned downstream of RepA box 4 (region II). Although DnaA was unable to unwind oriB on its own, it enhanced the RepA-induced KMnO4 reactivity of region II, as shown by the appearance of modified bases at position T1988, T1990, and T1991 of the top strand (Fig. 2A, lane 5) and T1996 of the bottom strand (Fig. 2B, lane 5), as well as the increased signal strength of modified bases.



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  		FIG. 2. Analysis of open complex formation by KMnO4 footprinting, using a supercoiled oriB template, as described in Materials and Methods. When present, RepA was added to 220 nM and DnaA was added to 40 nM. A+G, Maxam-Gilbert sequencing reaction; TGCA, sequencing ladder generated by using dideoxynucleotide terminators and T7 DNA polymerase. Residues sensitive to oxidation by KMnO4 are indicated.



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  		FIG. 3. (A) Sequence of the oriB fragment, showing residues sensitive to oxidation by KMnO4. The DnaA R box is boxed, the RepA boxes are indicated by lines below the sequence, and the 6-mer sequence is overlined. The DNase I-hypersensitive sites are indicated by arrows; residues modified by KMnO4 are shown in boldface, with sites of reactivity induced by RepA alone indicated by open stars; and those with increased reactivity upon addition of DnaA are indicated by solid stars. Points of insertion in the spacing mutants are indicated by triangles, with the number of base pairs inserted shown. The sequence of the scrambled RepA box 1 is shown in parentheses above the box (nucleotides in boldface are conserved in all boxes, and the substitutions are shown in lowercase). (B) Alignment, using Clustal W, of the first 129 bp of oriB of pMU720, with the corresponding sequences of pMU604 and pSW800. Nucleotides conserved in all three sequences are indicated by stars below the sequence. The DnaA R box of oriB is boxed, the 6-mer important for origin opening is in boldface, and the RepA boxes are indicated.

Importance of the sequence motif 5'-TCTTAA-3' in localized unwinding of oriB. Examination of the oriB sequence revealed that the KMnO4-sensitive region II contains the 6-mer sequence 5'-TCTTAA-3' (Fig. 3A), which is conserved in the origin of replication of pMU604 and pSW800, two plasmids distantly related to pMU720 (Fig. 3B). To investigate the importance of this 6-mer in open complex formation in vitro and plasmid replication in vivo, a mutant in which this sequence was replaced by 5'-cgaTcg-3' (substituted nucleotides are lowercase) was generated and examined. DNase I footprinting showed that this mutation did not alter the binding of DnaA and RepA to oriB (Fig. 4A). However, the mutation severely weakened the origin opening in region II, without affecting the KMnO4 sensitivity of region I (Fig. 4B). The loss of sensitivity to KMnO4 in the mutant oriB was not due to the absence of pyrimidines in region II, as there are T residues at positions 1988, 1990, and 1994 and a C at position 1991 on the top strand and T residues at positions 1987, 1989, and 1993 and C residues at positions 1992 and 1996 on the bottom strand. This mutant was unable to replicate in vivo. To determine whether the orientation of the 6-mer sequence, 5'-TCTTAA-3', was important to origin function, a mutant in which this sequence was reversed was constructed. This mutant too was unable to replicate in vivo. Thus, AT richness of the 6-mer sequence was not sufficient for origin activity, which required the presence of the 6-mer sequence in its native orientation.



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  		FIG. 4. (A) DNase I footprinting of the oriB fragment carrying mutated 6-mer sequence. Double-stranded oriB fragment, 5'-end-labeled in the bottom strand, was incubated with RepA and DnaA protein(s) and subjected to partial digestion with DNase I. RepA was added to 720 nM, and DnaA was added to 60 nM. A+G, Maxam-Gilbert sequencing reaction. Asterisks indicate sites of enhanced cleavage by DNase I. DB, DnaA R box; RB1 to RB4, Rep A boxes. (B) KMnO4 footprinting of oriB fragment with the mutated 6-mer sequence. RepA was added to 440 nM, and DnaA was added to 40 nM. Regions I and II of KMnO4 sensitivity in the wild-type oriB are indicated. A+G, Maxam-Gilbert sequencing reaction.

Importance of native positioning of the RepA boxes to localized unwinding of oriB. Previous studies have shown that binding of RepA to RepA boxes 1 and 2 promotes the appearance of hypersensitive bands at position 1951 of the top strand and positions 1949 and 1950 in the bottom strand, which is indicative of change in the local conformation of the DNA (2). Furthermore, although moving RepA boxes 1 and 2 closer together or further apart did not affect binding of RepA to the RepA boxes, it inhibited the appearance of the hypersensitive bands and had a severe deleterious effect on the ability of the mutant plasmids to replicate in vivo (2), suggesting that the conformational change induced by RepA may be important for an initiation step subsequent to initiator binding. To determine whether this step was origin opening, KMnO4 sensitivity of a mutant whose RepA box 1 had been scrambled and one carrying a 5-bp insertion between RepA boxes 1 and 2 was examined. Scrambling of RepA box 1, which abolishes binding of RepA to this box (2), had no detectable effect on KMnO4 sensitivity of region II but completely abolished the appearance of KMnO4-modified bases in region I (Fig. 5, lane 5). Addition of DnaA to the binding reaction did not increase susceptibility of region I to KMnO4 (Fig. 5, lane 6). Insertion of 5 bp between RepA boxes 1 and 2 significantly weakened the sensitivity of regions I and II to KMnO4 (Fig. 5, lane 8). Addition of DnaA to the binding reaction restored accessibility of region II to KMnO4, without significantly affecting region I (Fig. 5, lane 9). To determine whether increasing spacing between RepA boxes 2 and 4 would also disrupt origin opening, 9 bp were inserted at position 1972 (Fig. 3). This insertion did not affect binding of RepA to the RepA boxes nor the appearance of the hypersensitive bands at positions 1949 and 1950 of the bottom strand of oriB (data not shown), had little effect on KMnO4 sensitivity of region I, but severely weakened opening of region II (Fig. 5, lane 11). Addition of DnaA to the binding reaction restored susceptibility of region II to KMnO4 (Fig. 5, lane 12). This mutant was unable to replicate in vivo. These data suggest that the conformational change signaled by the appearance of hypersensitive bands at positions 1949 to 1951may be important for KMnO4 sensitivity of region I, but not that of region II, whereas the spacing between RepA boxes may be important for localized opening of region II, but not of region I. DnaA appears to be able to compensate for the defect in the opening of region II brought about by the inappropriate positioning of the RepA boxes but cannot correct the defect in the opening of region I. Furthermore, binding of RepA to RepA box 1, which is essential for replication in vivo (2), is not required for the opening of region II.



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  		FIG. 5. KMnO4 footprinting (bottom strand) of wild-type and mutant oriB fragments. RepA was added to 440 nM, and DnaA was added to 40 nM. Regions I and II of KMnO4 sensitivity in the wild-type oriB are indicated. A+G, Maxam-Gilbert sequencing reaction of the wild-type oriB; RB1, RepA box 1; RB1/2{Omega}5, oriB with a 5-bp insertion between RepA boxes 1 and 2; RB2/4{Omega}9, oriB with a 9-bp insertion between RepA boxes 2 and 4.


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DISCUSSION
 
Origin unwinding, a critical early step in replication initiation of most origins, is effected, at least in part, through stress placed on the structure of the ori by the binding of the initiator proteins to their specific recognition sequences. The unwound region usually lies close to, but doesn't overlap, the region where the initiator binds, makes both DNA strands accessible for modification by single-strand-specific agents, and is characterized by its AT richness. These traits are shared by one of the two KMnO4-sensitive regions of oriB, region II, which lies immediately downstream of RepA box 4 (the distal-most box), shows modification of all nine T residues on both strands of its 10-bp sequence, and has a G+C content of 10%. On the other hand, KMnO4-sensitive region I lies within the initiator-binding region, between RepA boxes 1 and 2, and shows modification of only two, adjacent, T residues on the bottom strand, despite the presence of six other T residues in the bottom strand and three in the top strand within the 15-bp spacer separating the two RepA boxes (Fig. 3A). Moreover, KMnO4 sensitivity of region I appears to be dependent on a conformational change in the DNA of the spacer region separating RepA boxes 1 and 2. These characteristics make it likely that region I represents a localized DNA distortion, which is known to cause KMnO4 sensitivity (4), rather than unwinding of the DNA helix.

The sequence (region II) opened as a consequence of initiator binding to oriB is 10 bp long, which represents one helical turn. This is considerably shorter than the regions of DNA opening in the origins of replication of some well-characterized plasmids of gram-negative bacteria that use the theta mode of replication, and of the E. coli chromosome. Thus, ~80 bp of the 112-bp AT-rich region in the {gamma} ori of plasmid R6K (27) and 46 bp of the ~60-bp AT-rich region in oriV of plasmid RK2 (23) are unwound. The DnaA-mediated unwinding of oriC extends over 28 bp in the absence and 54 bp in the presence of Eco SSB protein (25, 26). The shortness of the unwound sequence may be a reflection of the size and positioning of the AT-rich region of oriB. Analysis of the G+C content of oriB shows that the repA-proximal half of oriB, which contains the DnaA and RepA boxes and the 11-bp AT-rich region, has a G+C content of ~34%, whereas the distal half of oriB has a G+C content that, at 48%, is not significantly different from that of the minimal replicon of pMU720 as a whole. Thus, the AT-rich region of oriB is bordered on one side by RepA box 4 and on the other side by a run of four C residues, both of which may act as barriers to the unwinding of the DNA helix in vitro. However, given that one DnaB hexamer binds a single-stranded region of 20 ± 3 nt (18), it is likely that the region of oriB opening in vivo is larger than that detected in the present study, as host proteins other than DnaA may also contribute to opening of oriB. One such protein, HU, is required for origin opening in plasmids P1 (38) and F (19).

Many prokaryotic origins of replication contain tandem repeats in their AT-rich regions (5). In E. coli, DnaA catalyzes opening of the oriC duplex at three tandem repeats of a 13-mer sequence bearing consensus 5'-GATCTnTTnTTTT-3'. Although the presence of only the right 13-mer was sufficient for open complex formation in vitro, efficient formation of the prepriming complex required the presence of all three 13-mers (5). Detailed mutational analysis indicated that oriC activity, both in vitro and in vivo, requires sequence specificity in the middle and right 13-mers, but only AT richness in the left 13-mer (16). The AT-rich regions in the origins of replication of several members of the iteron-regulated family of plasmids also contain 13-mers whose sequences resemble those of the 13-mers present in oriC. Thus, pSC101 has two tandem 13-mers matching the consensus of those in oriC (5); F has one 13-mer, which has a single mismatch with the oriC consensus (19); and RK2 has four 13-mers bearing the consensus 5'-TAAACnTTnTTTT-3' (23). The AT-rich region of oriB of pMU720, which is not an iteron-regulated plasmid but a member of the extended family of FII/I complex group of plasmids, contains a 6-mer 5'-TCTTAA-3' that is conserved in the origins of replication of plasmids distantly related to pMU720 (2). In pMU720, this 6-mer is important for both origin opening in vitro and origin activity in vivo, and given the conservation of both its sequence and location, it is highly likely that it plays an equivalent role in replication of pMU604 and pSW800. It is striking that the sequence similarity between the oris of pMU720, pMU604, and pSW800 terminates immediately downstream of the 6-mer (Fig. 3B). The minimal ori of pMU720 has been defined and has been shown to extend 81 bp downstream of the 6-mer (41), but no function has been assigned to this region. It is noteworthy that despite its dissimilarity, the equivalent sequence from ori of pMU604 could replace the last 81 bp of oriB, with the resultant ori retaining 60% of wild-type activity (L. Borrell and J. Praszkier, unpublished data).

Although DnaA and RepA showed mutual cooperativity in their binding to oriB, DnaA could bind to the single DnaA R box in oriB independently of RepA. In contrast, DnaA failed to bind to the DnaA R box in the ori of the IncFII plasmid R1 in the absence of RepA, despite the sequences of the DnaA R boxes of the two plasmids being identical (32). This difference in the binding affinity supports the conclusion drawn from analyzing efficiency of binding of DnaA to single DnaA R boxes from oriC, located on 21-mer oligonucleotides and flanked either by their native or altered sequences, that DNA context influences the strength of binding of DnaA to its cognate binding site (45). DnaA appeared to play an auxiliary role in localized unwinding of oriB, augmenting the function of RepA without itself being able to open this origin. In this respect, the role of DnaA in origin opening of oriB was similar to that played by this protein in opening the origins of plasmids RK2 (23) and F (19). However, whereas replication of RK2 and F is absolutely dependent on DnaA (11, 21), pMU720 can replicate in a dnaA-null host (Borrell and Praszkier, unpublished). The finding that deleting the DnaA R box or moving it half a helical turn with respect to the RepA boxes reduces the copy number of plasmids replicating from oriB suggests that DnaA does play a role in replication of pMU720 in a wild-type host and that this role may require interaction between ori-bound RepA and DnaA (40, 41). Thus, pMU720 appears to be similar to the IncFII plasmids R1 and R100, to which it is phylogenetically related (36), in that all three plasmids can replicate in the absence of DnaA, but less efficiently than when this protein is present (35, 52).

The positioning of the RepA boxes with respect to one another was important for opening of oriB, as moving the boxes apart severely reduced the unwinding activity of RepA. Plasmids carrying these insertion mutations were unable to replicate in vivo, despite the fact that addition of DnaA restored open complex formation in vitro. Similarly, binding of RepA to RepA box 1, which is essential to oriB activity in vivo (2), was not required for origin opening in vitro. These findings suggest that binding of RepA to correctly positioned RepA boxes may be important not only for the opening of oriB, but also for a subsequent step in the initiation pathway, and that DnaA is unable to compensate for a defect caused by incorrectly positioned RepA molecules. This step is likely to be loading and activation of DnaB helicase to the opened oriB. In plasmid RK2, recruitment of DnaB to oriV is carried out by DnaA, which delivers the DnaBC complex to the cluster of four DnaA R boxes located ~200 bp from the region of TrfA-mediated origin opening (37). Repositioning of DnaBC to the opened region of oriV and activation of DnaB require interaction with TrfA and precise positioning of DnaA R boxes (10, 24). Thus, although moving the DnaA R boxes by 6 bp had no effect on open complex formation, it severely reduced loading of the DnaB helicase (10). A similar requirement for positioning of RepA and DnaA R boxes may operate in oriB.


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ACKNOWLEDGMENTS
 
This work was supported by a grant from the National Health and Medical Research Council.

We are grateful to Aresa Toukdarian and Donald Helinski for providing the strain for purification of DnaA.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia. Phone: 61 3 8344 7751. Fax: 61 3 9347 1540. E-mail: judy{at}ariel.its.unimelb.edu.au. Back


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Journal of Bacteriology, June 2004, p. 3785-3793, Vol. 186, No. 12
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.12.3785-3793.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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