The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

Molecular structure of the replication origin of a Bacillus amyloliquefaciens plasmid pFTB14

Summary The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin for the plasmid upstream of the promoter. The rep product is trans-active and essential for plasmid replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB110 and protein A of pC194 (all these found in staphylococci) and the protein of the R6K plasmid of Escherichia coli. The predicted rep protein has highly homologous amino acid sequences with protein A of pC194 and RepC of pUB110 throughout the protein molecule, but not with RepC of pT181, of R6K or protein RepH encoded by and iniating the replication of pC194.     

Conformational changes induced by integration host factor at origin gamma of R6K and copy number control.

We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number. Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites. The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding. Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding. Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry. The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence. We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.     

Activation of distant replication origins in vivo by DNA looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance.

We have isolated mutants of the pi initiator protein of the plasmid R6K that are defective in DNA looping in vitro but retain their normal DNA binding affinity for the primary binding sites (iterons) at the gamma origin/enhancer. One such looping defective mutant called R6 was determined to be a proline to leucine change at position 46 near the N terminus of the pi protein. Using a set of genetic assays that discriminate between the activation of the gamma origin/enhancer from those of the distantly located alpha and beta origins, we show that the looping defective initiator protein fails to activate the alpha and beta origins but derepresses initiation from the normally silent gamma origin in vivo. The results conclusively prove that DNA looping is required to activate distant replication origins located at distances of up to 3 kb from the replication enhancer.     

Enhancer-origin interaction in plasmid R6K involves a DNA loop mediated by initiator protein

Initiation of DNA replication from ori beta of plasmid R6K requires the presence of the ori gamma sequence in cis. We demonstrate that binding of initiator protein to the seven strong, tandem binding sites in gamma increases binding of the protein at the very weak binding site present in ori beta by cooperativity at a distance. The gamma-beta interaction via the initiator results in a DNA loop, as revealed by the novel technique of cyclization enhancement and as confirmed by exonuclease III protection, electron microscopy, and chemical footprinting. The protein-mediated gamma-beta interaction in vitro suggests that the cooperative interaction of gamma-bound protein with the beta sequence by DNA looping is an early step in the initiation of DNA replication at the beta origin of R6K.     

Activation in vivo of the minimal replication origin beta of plasmid R6K requires a small target sequence essential for DNA looping.

The plasmid R6K contains three distinct origins of replication: alpha, beta, and gamma. The gamma sequence is essential in cis and acts as an enhancer that activates the distant alpha and beta origins. R6K therefore represents a favorable procaryotic model system with which to unravel the biochemical mechanisms underlying selective origin activation, particularly activation involving distant sites on the same chromosome. We have discovered that plasmids containing the origins alpha and gamma required the Escherichia coli DnaA initiator protein in addition to the R6K-encoded initiator protein, Pi, and other host replisomal proteins for their maintenance in vivo. Plasmids initiating replication from origin beta required only the Pi initiator protein and other host replisomal proteins. We have exploited the differential requirement for the DnaA protein by origins gamma and beta to selectively study and localize the minimal origin beta sequences by deletion analysis as one test of a looping model of origin activation. A 64-bp region spanning the extreme -COOH terminal coding sequence of the Pi protein was found to be essential for replication in vivo in the absence of DnaA protein, consistent with the approximate physical location of the beta origin. Replication emanating from origin beta could be abolished in vivo by deletion of the 9-bp target site for Pi protein-mediated DNA looping between the gamma origin/enhancer and the distant beta origin. Electron microscopy of nascent replication intermediates generated in vivo directly confirmed our genetic localization of the beta origin. Our results strongly suggest that activation of the beta origin by a distant replication enhancer element requires a small target sequence essential for initiator protein-mediated DNA looping.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage φX174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5′ region has been identified and is functional in Bacillus subtilis.     

Control of plasmid R6K copy numbers in isogenic rep+ and rep Escherichia coli strains

Summary We have analysed as a function of cell doubling times the control of R6K plasmid replication in rep ⁺ and rep strains of Escherichia coli. The rep mutation results in an alteration or loss of an enzyme that unwinds helical DNA. We found in rep ⁺ bacteria that R6K relative dosage (plasmids per genome equivalent) remained nearly constant as growth rates increased. From this we concluded that the average plasmid concentration (plasmids per unit cell mass or volume) fell relative to the average concentration of chromosome origins when growth rates increased. In this context, the control of R6K replication is similar to that of other plasmids as seen by different workers. We also found that the relative dosage of R6K in rep mutants is greater than in rep ⁺ bacteria when both strains were grown at fast growth rates. This finding was expected since at fast growth rates the number of genome equivalents per unit mass is expected to be lower in rep mutants. Unexpectedly, however, we found the effect of the rep mutation on R6K relative dosage had occurred in a step-like manner at a slow growth rate of about 120 min per generation. This implies that both the relative dosage and concentration of R6K had increased in a step-like manner. We also found that the effect of the rep mutation on R6K concentration was lost at fast growth rates while the effect of the mutation on R6K relative dosage was not lost.     

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.     

The integration host factor of Escherichia coli binds to multiple sites at plasmid R6K gamma origin and is essential for replication.

Examination of the effect of the himA and himD mutants of E. coli on the maintenance of plasmid R6K has revealed that the gamma origin-containing replicons cannot be established in any of the mutants deficient in the production of E. coli Integration Host Factor (IHF). Contrary, the R6K derivatives containing other origins of the plasmid (alpha and/or beta) replicate in a host lacking functional IHF protein. We show that IHF protein binds specifically to a segment of the replication region which is essential for the activity of all three R6K origins. Mapping the IHF binding sequence with neocarzinostatin showed that the protein protects three segments of the origin: two strong binding sites reside within an AT-rich block, while the third, considerably weaker site is separated from the other two by a cluster of the seven 22 bp direct repeats. These seven repeats have been shown previously to bind the R6K-encoded initiator protein pi. We also demonstrate that the establishment of pi-origin complexes prior to IHF addition prevents the binding of the IHF protein to the gamma origin. The binding sequences of IHF and pi proteins do not overlap, therefore, we propose that the binding of pi protein alters the structure of the DNA and thereby prevents the subsequent binding of IHF protein.     

Gene encoding a replication initiator protein and replication origin of conjugative plasmid pSA1.1 of Streptomyces cyaneus ATCC 14921

pSA1.1 is a 9.1-kb multicopy plasmid originally isolated from Streptomyces cyaneus (formerly S. azureus) ATCC 14921. This plasmid accumulates single-stranded DNA in S. lividans and is therefore considered to replicate by a rolling-circle replication. In the present work, the rep gene encoding the replication initiator protein and the replication origin ori of pSA1.1 were determined. The rep and ori are located on separate regions. The Rep protein of pSA1.1 belongs to superfamily I which includes A proteins of phages. Nucleotide sequence of the surrounding putative nicking site of pSA1.1 shows good agreement with those of the pC194 group plasmids and phages. The direction of replication was also determined.     

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).     

Role of individual monomers of a dimeric initiator protein in the initiation and termination of plasmid rolling circle replication

Plasmids of the pT181 family encode initiator proteins that act as dimers during plasmid rolling circle (RC) replication. These initiator proteins bind to the origin of replication through a sequence-specific interaction and generate a nick at the origin that acts as the primer for RC replication. Previous studies have demonstrated that the initiator proteins contain separate DNA binding and nicking-closing domains, both of which are required for plasmid replication. The tyrosine residue at position 191 of the initiator RepC protein of pT181 is known to be involved in nicking at the origin. We have generated heterodimers of RepC that consist of different combinations of wild type, DNA binding, and nicking mutant monomers to identify the role of each of the two monomers in RC replication. One monomer with DNA binding activity was sufficient for the targeting of the initiator to the origin, and the presence of Tyr-191 in one monomer was sufficient for the initiation of replication. On the other hand, a dimer consisting of one monomer defective in DNA binding and the other defective in origin nicking failed to initiate replication. Our results demonstrate that the monomer that promotes sequence-specific binding to the origin must also nick the DNA to initiate replication. Interestingly, whereas Tyr-191 of the initiator was required for nicking at the origin to initiate replication, it was dispensable for termination, suggesting that alternate amino acids in the initiator may promote termination but not initiation.     

Conformational changes in a replication origin induced by an initiator protein

The replication initiator protein of the plasmid R6K binds to seven contiguous 22 bp direct repeats that form an indispensable part of the three replication origins alpha, beta, and gamma. Binding of the initiator to the direct repeats induced a marked bending of the region of gamma replication origin. Binding of the initiator also promoted unwinding of the origin DNA by at least two turns. Distamycin appeared to antagonize the binding of the initiator to the seven 22 bp direct repeats. At the appropriate DNA and protein concentrations the initiator enhanced topoisomerase-induced catenation of the origin containing supercoiled DNA but not of DNA lacking the origin sequence. Thus, the initiator protein caused significant changes in the secondary and tertiary structures of the replication origin.     

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.     

Autogenous regulation of synthesis of the replication protein in plasmid pSC101

Summary A 1.3-kb segment of plasmid pSC101 includes the replication origin (ori) and the gene (rep) encoding the 37 kilodalton (K) protein required for autonomous replication of the plasmid. The present work describes the regulation of the rep gene expression. The promoters P _R and P _L fail to promote rep gene expression when located upstream of a sequence with dyad symmetry overlapping the rep promoter, whereas elimination of this sequence allows expression and results in over-production of the rep protein. When expression of trpA-lacZ is controlled under the rep promoter, -galactosidase is produced without the lac inducer. However, this enzyme synthesis is effectively reduced when the complete rep sequence is provided in trans. A partial disruption of the sequence with dyad symmetry relieves the repression. These results suggest that expression of the rep gene is negatively regulated by its own product and that the sequence with dyad symmetry plays the role of a receptor site for the rep protein.Abbreviations bp base pairs - kb kilobase pairs - ORF open reading frame - SDS sodium dodecyl sulphate - ts temperature sensitive     

Integration host factor of Escherichia coli reverses the inhibition of R6K plasmid replication by pi initiator protein.

Integration host factor (IHF) protein is the only host-encoded protein known to bind and to affect replication of the gamma origin of Escherichia coli plasmid R6K. We examined the ability of R6K origins to replicate in cells lacking either of the two subunits of IHF. As shown previously, the gamma origin cannot replicate in IHF-deficient cells. However, this inability to replicate was relieved under the following conditions: underproduction of the wild-type pi replication protein of R6K or production of normal levels of mutant pi proteins which exhibit relaxed replication control. The copy number of plasmids containing the primary R6K origins (alpha and beta) is substantially reduced in IHF-deficient bacteria. Furthermore, replication of these plasmids is completely inhibited if the IHF-deficient strains contain a helper plasmid producing additional wild-type pi protein. IHF protein has previously been shown to bind to two sites within the gamma origin. These sites flank a central repeat segment which binds pi protein. We propose a model in which IHF binding to its sites reduces the replication inhibitor activity of pi protein at all three R6K origins.     

Specificity of RepC protein in plasmid pT181 DNA replication

The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 DNA replication specifically requires the plasmid-encoded initiator protein, RepC. The initiator protein binds specifically to a 32-base pair sequence within the pT181 origin of replication. RepC protein also has a nicking-closing activity that is specific for the pT181 origin. Replication of pT181 initiates by covalent extension of the nick and proceeds by a rolling circle mechanism. Two other small, multicopy plasmids pC221 and pS194 belong to the pT181 family and have common structural organization and replication properties. The replication proteins and replication origins of these plasmids have extensive sequence homologies, although they belong to different incompatibility groups. In spite of this homology, the replication proteins and replication origins of these three plasmids do not show any cross-reactivity in vivo. We have carried out a series of in vitro experiments to determine the specificity of pT181-encoded initiator protein, RepC. DNA binding experiments showed that although the binding of RepC to the pT181 origin was very efficient, little or no binding was seen with pC221 and pS194 origins. The nicking-closing activity of RepC was found to be equally efficient with the pC221 and pS194 plasmids. The plasmids pC221 and pS194 replicated efficiently in a RepC-dependent in vitro system. However, replication of these plasmids was greatly reduced in the presence of a competing pT181 origin. The results presented here suggest that nicking-closing by RepC at the origin is not sufficient for maximal replication and that tight binding of RepC to the origin plays an important role in the initiation of DNA replication.     

Roles of a 106-bp origin enhancer and Escherichia coli DnaA protein in replication of plasmid R6K.

A dnaA 'null' strain could not support replication of intact plasmid R6K or derivatives containing combinations of its three replication origins (alpha, gamma, beta). DnaA binds in vitro to sites in two functionally distinct segments of the central gamma origin. The 277-bp core segment is common to all three origins and contains DnaA box 2, which cannot be deleted without preventing replication. Immediately to the left of the core lies the 106-bp origin enhancer, which contains DnaA box 1. When the origin enhancer is deleted, the core alone can still initiate replication if levels of wt pi protein are decreased or if copy-up pi mutant proteins are provided in trans. DnaA does not effect expression of R6K replication initiator protein pi, although several DnaA boxes were identified in the coding segment of the pir gene, which encodes pi. Together these data suggest that a single DnaA box, 2, is sufficient for initiation from the gamma origin and might be sufficient for initiation from the gamma origin and might be sufficient and required for the activity of the alpha and beta origins as well. Implications of the DnaA protein binding to two domains of the gamma origin and the role of the 106-bp origin enhancer in replication are discussed.     

Specificity of the interactions between the Rep proteins and the origins of replication of Staphylococcus aureus plasmids pT181 and pC221

Summary pT181 and pC221 are closely relatedStaphylococcus aureus plasmids with the same genome organization, which is characterized by the overlapping of the origin of replication with the sequence encoding a protein, Rep, essential for plasmid replication. Former results have shown the lack of in vivo cross-complementation between these two plasmids, while in vitro studies have revealed the ability of both Rep proteins to act on either origin. One possible explanation for this difference was based on a previous analysis of the incompatibility expressed by the origin of replication of these plasmids, showing that the origin embedded in therep gene competes for Rep utilization with the origin of a test plasmid and that changes in the sequence of the origin reduce its ability to compete. To avoid this problem, in the present work special hybrids were constructed in which the origin of replication overlapping therep gene was mutationally inactivated, without changing the amino acid sequence of the encoded protein. The level of Rep expression by these hybrids could be varied by taking advantage of what is presently known about the control of Rep synthesis in plasmid pT181. The results of complenentation studies conducted using these hybrids have shown that: (i) at the usual level of expression for a wild-type plasmid each Rep protein can initiate replication strictly from its corresponding origin; (ii) when overproduced, the pT181 RepC protein could also act efficiently on the pC221 origin; a functional pT181 origin present in the same host completely prevented this complementation; (iii) in excess, the RepD protein encoded by pC221 could replicate a plasmid carrying the pT181 origin but could not ensure the hereditary stability of such a plasmid in the absence of another active replication system; (iv) when overproduced both RepC and RepD could act on the origin of replication of three other related plasmids pS194, pC223 and pUB112.