DnaA dependent replication of plasmid R1 occurs in the presence of point mutations that disrupt the dnaA box of oriR.

We have found that DnaA dependent replication of R1 still occurred when 5 of the 9 bases in the dnaA box present in oriR were changed by site directed mutagenesis although the replication efficiency decreased to 20% and 70% of the wild-type origin in vitro and in vivo respectively. Additional mutation of a second dnaA box, 28 bp upstream oriR, that differs in only one base from the consensus sequence, did not affect the level of replication whereas polyclonal antibodies against DnaA totally abolished in vitro replication in the absence of the dnaA box. Wild-type RepA as well as a RepA mutant, RepA2623, that binds to oriR but that is inactive in promoting in vitro replication of plasmid R1, induce efficient binding of DnaA to the dnaA box. However, specific binding of DnaA to oriR was not detected by DNase I protection experiments in the absence of the dnaA box. These results suggest that the entrance of the DnaA protein in oriR is promoted initially by interactions with a RepA-oriR pre-initiation complex and that, in the absence of the dnaA box, these interactions can support, with reduced efficiency, DnaA dependent replication of plasmid R1.     

P1 plasmid replication. Purification and DNA-binding activity of the replication protein RepA

The minimal P1 replicon encompasses an open reading frame for the essential replication protein, RepA, bracketed by two sets of multiple 19-base pair repeated sequences, incA and incC. This study focused on the interaction of RepA with the incC and incA repeated sequences because earlier studies suggested that incA might control P1 copy number by titrating limiting amounts of RepA and because the incC repeats, which are part of the origin of replication, contain the promoter for repA. RepA is essential for origin function, autoregulates its own synthesis from the promoter, and, when overproduced, blocks origin function. In this study, RepA was overproduced from an expression vector and purified to 90% homogeneity. The binding of RepA to the DNA encompassing repeat sequences was assayed by monitoring the mobility of protein-DNA complexes on polyacrylamide gels. Distinct species of retarded bands were seen with the maximum number of bands corresponding to the number of repeats present in the target fragment. No evidence was found for RepA binding to fragments not containing the repeats. This suggests that the specific binding of RepA to the repeats may be involved in each of the diverse activities of RepA.     

Role of RepA and DnaA proteins in the opening of the origin of DNA replication of an IncB plasmid

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.     

Deletion analysis of the mini-P1 plasmid origin of replication and the role of Escherichia coli DnaA protein

The mini-P1 plasmid origin of replication is contained on a 246 base pair (bp) piece of DNA. At one end there are five 19-bp binding sites for the P1 initiator protein, RepA, and near the other end there are two 9-bp DnaA protein-binding sites. To further define the limits of the origin, we cloned the origin region in M13 and constructed deletions of either end. We sequenced the DNA and tested the replicative form I DNA of the deletion phages for their ability to support RepA-dependent DNA replication in an in vitro system. The origin that is functional in vitro could be reduced to 202 bp. It includes three intact and one incomplete RepA-binding sites at one end and the two DnaA-binding sites at the other end. When the two naturally occurring DnaA-binding sites were replaced with one or two synthetic sites, only the construction containing two sites was active in vitro. We found that the minimal origin that is functional in vivo contains all of the five RepA and the two DnaA-binding sites. Mini-P1 plasmid replication both in vivo and in vitro requires two initiator proteins, the Escherichia coli DnaA protein and the P1 RepA protein. We have found that the ADP form of DnaA is as active as the ATP form of the protein in the in vitro replication of mini-P1. In contrast, only the ATP form is active for in vitro replication of plasmids carrying the E. coli origin (Bramhill, D., and Kornberg, A. (1988) Cell 52, 743-755).     

Interaction of initiator proteins with the origin of replication of an IncL/M plasmid

The origin of replication of the IncL/M plasmid pMU604 was analyzed to identify sequences important for binding of initiator proteins and origin activity. A thrice repeated sequence motif 5'-NANCYGCAA-3' was identified as the binding site (RepA box) of the initiator protein, RepA. All three copies of the RepA box were required for in vivo activity and binding of RepA to these boxes appeared to be cooperative. A DnaA R box (box 1), located immediately upstream of the RepA boxes, was not required for recruitment of DnaA during initiation of replication by RepA of pMU604 unless a DnaA R box located at the distal end of the origin (box 3) had been inactivated. However, DnaA R box 1 was important for recruitment of DnaA to the origin of replication of pMU604 when the initiator RepA was that from a distantly related plasmid, pMU720. A mutation which scrambled DnaA R boxes 1 and 3 and one which scrambled DnaA R boxes 1, 3 and 4 had much more deleterious effects on initiation by RepA of pMU720 than on initiation by RepA of pMU604. Neither Rep protein could initiate replication from the origin of pMU604 in the absence of DnaA, suggesting that the difference between them might lie in the mechanism of recruitment of DnaA to this origin. DnaA protein enhanced the binding and origin unwinding activities of RepA of pMU604, but appeared unable to bind to a linear DNA fragment bearing the origin of replication of pMU604 in the absence of other proteins.     

The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA.

The in vitro replication of R1 miniplasmid promoted by purified preparations of the plasmid encoded RepA protein in cell extracts of E. coli is resistant to rifampicin and can be completely inhibited by antibodies against DnaG, the primase of the cell, as well as by antibodies against proteins DnaB and SSB. R1 replication is abolished in extracts deficient in the DnaA protein. This deficiency is efficiently complemented by purified preparations of the DnaA protein. The in vitro replication of plasmid R1 is also abolished in DnaC deficient extracts and by a 10 bp deletion (nucleotides 1463-1472) within the minimal origin region. These data indicate the requirement of the DnaA, DnaB, DnaC, DnaG and SSB replication proteins of the host, as well as of specific oriR1 sequences for the RepA dependent replication of plasmid R1. The implications of these results for the initiation of R1 replication are discussed.     

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

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

A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene.

The P1 plasmid replication origin P1oriR is controlled by methylation of four GATC adenine methylation sites within heptamer repeats. A comparable (13mer) region is present in the host origin, oriC. The two origins show comparable responses to methylation; negative control by recognition of hemimethylated DNA (sequestration) and a positive requirement for methylation for efficient function. We have isolated a host protein that recognizes the P1 origin region only when it is isolated from a strain proficient for adenine methylation. The substantially purified 22 kDa protein also binds to the 13mer region of oriC in a methylation-specific fashion. It proved to be the product of the seqA gene that acts in the negative control of oriC by sequestration. We conclude that the role of the SeqA protein in sequestration is to recognize the methylation state of P1oriR and oriC by direct DNA binding. Using synthetic substrates we show that SeqA binds exclusively to the hemimethylated forms of these origins forms that are the immediate products of replication in a methylation-proficient strain. We also show that the protein can recognize sequences with multiple GATC sites, irrespective of the surrounding sequence. The basis for origin specificity is primarily the persistence of hemimethylated forms that are over-represented in the natural. DNA preparations relative to controls.     

Boundaries of the pSC101 minimal replicon are conditional.

The DNA segment essential for plasmid replication commonly is referred to as the core or minimal replicon. We report here that host and plasmid genes and sites external to the core replicon of plasmid pSC101 determine the boundaries and competence of the replicon and also the efficiency of partitioning. Missense mutations in the plasmid-encoded RepA protein or mutation of the Escherichia coli topoisomerase I gene enable autonomous replication of a 310-bp pSC101 DNA fragment that contains only the actual replication origin plus binding sites for RepA and the host-encoded DnaA protein. However, in the absence of a repA or topA mutation, the DNA-bending protein integration host factor (IHF) and either of two cis-acting elements are required. One of these, the partitioning (par) locus, is known to promote negative DNA supercoiling; our data suggest that the effects of the other element, the inverted repeat (IR) sequences that overlap the repA promoter, are mediated through the IR's ability to bind RepA. The concentrations of RepA and DnaA, which interact with each other and with plasmid DNA in the origin region (T. T. Stenzel, T. MacAllister, and D. Bastia, Genes Dev. 5:1453-1463, 1991), also affect both replication and partitioning. Our results, which indicate that the sequence requirements for replication of pSC101 are conditional rather than absolute, compel reassessment of the definition of a core replicon. Additionally, they provide further evidence that the origin region RepA-DnaA-DNA complex initiating replication of pSC101 also mediates the partitioning of pSC101 plasmids at cell division.     

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

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

Footprinting studies of specific complexes formed by RepA, a replication initiator of plasmid pCU1, and its binding site

The basic replicon of plasmid pCU1 contains three different replication origins. Replication initiated from the oriB origin requires pCU1-encoded protein RepA. Previously, information analysis of 19 natural RepA binding sequences predicted a 20-bp sequence as a RepA binding site. Guanines contacting RepA in the major groove of DNA have also been determined. In this study, we used the missing-nucleoside method to determine all of the bases relevant to RepA binding. The importance of some thymine bases was also confirmed by a missing-thymine site interference assay. Participation of the 5-methyl groups of two thymines (at positions -6 and 7) in RepA binding was pointed out by a missing-thymine methyl site interference assay. Phosphate groups of the DNA backbone which strongly interfered with RepA binding upon ethylation were also identified. The pattern of contacting positions mapped by hydroxyl radical protection footprinting indicates that RepA binds to one face of B-form DNA. The length of the binding site was found to be 20 bp by dissociation rate measurement of complexes formed between RepA and a variety of binding sequences. The symmetry of the binding site and that of the contacting bases, particularly the reacting 5-methyl groups of two thymines, suggest that pCU1-encoded RepA may contact its site as a homodimer.     

Control of P1 plasmid replication by iterons

The incA locus of plasmid P1 controls plasmid copy number by inhibiting the replication origin, oriR . Both loci contain repeat sequences (iterons) that bind the P1 RepA protein. Regulation appears to occur by contact of incA and oriR loci of daughter plasmids mediated by RepA-bound iterons. Synthetic incA iteron arrays were constructed with altered numbers, sequences or spacing of iterons. Using these in in vitro and in vivo assays, we examined two models: (i) that the origin and incA loci form a stable 1:1 complex in which multiple iterons of each locus are paired with those of the other, and (ii) that individual incA iterons act as freely diffusing nucleoprotein units that contact origin iterons in a random and dynamic fashion. The data presented here strongly favour the latter case. The origin, with its five iterons, acts as a target but not as an effector of regulation. We present a model for replication control based on random, dynamic contacts between incA iterons and the origin. This system would display randomness with respect to choice of templates and timing of initiation if multiple replicon copies were present, but would tend to act in a machine-like fashion in concert with the cell cycle if just two copies were present in a dividing cell.     

Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM beta 1 and pSM19035.

The large conjugative plasmid pIP501 was originally isolated from Streptococcus agalactiae. To study the molecular basis of pIP501 replication we determined the nucleotide sequence of a 2.2 kb DNA segment which is essential and sufficient for autonomous replication of pIP501 derived plasmids, in Bacillus subtilis cells. This region can be divided into two functionally discrete segments: a 496 bp region (oriR) that acts as an origin of replication, and a 1488 bp segment coding for an essential replication protein (RepR). The RepR protein, which has a molecular mass of 57.4 kDa, could complement in trans a thermosensitive replicon bearing the pIP501 origin. Chimeric Rep proteins and replicons were obtained by domain swapping between rep genes of closely related streptococcal plasmids belonging to the inc18 group (pIP501, pAM beta 1 and pSM19035). The chimeras were functional in B. subtilis.     

Isolation and characterization of a conditional replication mutant of the antibiotic resistance factor R1 affected in the gene of the replication protein RepA

Summary In vitro mutagenesis with hydroxylamine of a ParD^- miniderivative of R1, pAB174, yielded mutants that were less stable in the cell than pAB174. Some of these mutants had a thermosensitive phenotype. The replication of pAB2623, one of the thermosensitive mutants, was inhibited in the cell at the restrictive temperature of 42° C. The efficiency of the RepA protein of pAB2623 to promote replication of R1 in an in vitro assay was greatly reduced. Sequence analysis indicated that the repA gene of pAB2623 contains, close to its 3 end, two GC-AT transitions, separated by a single base, that change two consecutive codons of the gene. These results indicate that the phenotype of the mutant is the consequence of a mutated RepA protein and is consistent with the requirement of RepA for the in vivo replication of this plasmid.     

The repA gene of the linear Yersinia enterocolitica prophage PY54 functions as a circular minimal replicon in Escherichia coli

The Yersinia enterocolitica prophage PY54 replicates as a linear DNA molecule with covalently closed ends. For replication of a circular PY54 minimal replicon that has been derived from a linear minireplicon, two phage-encoded loci are essential in Escherichia coli: (i) the reading frame of the replication initiation gene repA and (ii) its 212-bp origin located within the 3' portion of repA. The RepA protein acts in trans on the origin since we have physically separated the PY54 origin and repA onto a two-plasmid origin test system. For this trans action, the repA 3' end carrying the origin is dispensable. Mutagenesis by alanine scan demonstrated that the motifs for primase and for nucleotide binding present in the protein are essential for RepA activity. The replication initiation functions of RepA are replicon specific. The replication initiation proteins DnaA, DnaG, and DnaB of the host are unable to promote origin replication in the presence of mutant RepA proteins that carry single residue exchanges in these motifs. The proposed origins of the known related hairpin prophages PY54, N15, and PKO2 are all located toward the 3' end of the corresponding repA genes, where several structure elements are conserved. Origin function depends on the integrity of these elements.     

Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin.

Binding of the P1-encoded protein RepA to the origin of P1 plasmid replication is essential for initiation of DNA replication and for autoregulatory repression of the repA promoter. Previous studies have shown defects in both initiation and repression in hosts lacking heat shock proteins DnaJ, DnaK, and GrpE and have suggested that these proteins play a role in the RepA-DNA binding required for initiation and repression. In this study, using in vivo dimethyl sulfate footprinting, we have confirmed the roles of the three heat shock proteins in promoting RepA binding to the origin. The defects in both activities could be suppressed by increasing the concentration of wild-type RepA over the physiological level. We also isolated RepA mutants that were effective initiators and repressors without requiring the heat shock proteins. These data suggest that the heat shock proteins facilitate both repression and initiation by promoting only the DNA-binding activity of RepA. In a similar plasmid, F, initiator mutants that confer heat shock protein independence for replication were also found, but they were defective for repression. We propose that the initiator binding involved in repression and the initiator binding involved in initiation are similar in P1 but different in F.