Structural changes in RepA,a plasmid replication initiator,upon binding to origin DNA

RepA protein is the DNA replication initiator of the Pseudomonas plasmid pPS10. RepA dimers bind to an inversely repeated operator sequence in repA promoter, thus repressing its own synthesis, whereas monomers bind to four directly repeated sequences (iterons) to initiate DNA replication. We had proposed previously that RepA is composed of two winged-helix (WH) domains, a structural unit also present in eukaryotic and archaeal initiators. To bind to the whole iteron sequence through both domains, RepA should couple monomerization to a conformational change in the N-terminal WH, which includes a leucine zipper-like sequence motif. We show for the first time that, by itself, binding to iteron DNA in vitro dissociates RepA dimers into monomers and alters RepA conformation, suggesting an allosteric effect. Furthermore, we also show that similar changes in RepA are promoted by mutations that substitute two Leu residues of the putative leucine zipper by Ala, destabilizing the hydrophobic core of the first WH. We propose that this mutant (RepA-2L2A) resembles a transient folding intermediate in the pathway leading to active monomers. These findings, together with the known activation of other Rep-type proteins by chaperones, are relevant to understand the molecular basis of plasmid DNA replication initiation.     

Interaction of the initiator protein of an IncB plasmid with its origin of DNA replication

The replication initiator protein RepA of the IncB plasmid pMU720 was purified and used in DNase I protection assays in vitro. RepA protected a 68-bp region of the origin of replication of pMU720. This region, which lies immediately downstream of the DnaA box, contains four copies of the sequence motif 5'AANCNGCAA3'. Mutational analyses identified this sequence as the binding site specifically recognized by RepA (the RepA box). Binding of RepA to the RepA boxes was ordered and sequential, with the box closest to the DnaA binding site (box 1) occupied first and the most distant boxes (boxes 3 and 4) occupied last. However, only boxes 1, 2, and 4 were essential for origin activity, with box 3 playing a lesser role. Changing the spacing between box 1 and the other three boxes affected binding of RepA in vitro and origin activity in vivo, indicating that the RepA molecules bound to ori(B) interact with one another.     

cmp, a cis-acting plasmid locus that increases interaction between replication origin and initiator protein.

pT181, a 4.4-kilobase multicopy plasmid of Staphylococcus aureus, encodes a trans-acting initiator protein, RepC, which was rate limiting for replication. Deletions in a 500-base-pair region of the plasmid external to the minimal replicon decreased the ability of the plasmid to compete with a coexisting incompatible plasmid. These deletions, which define a region called cmp (for competition), appeared to affect the interaction of RepC and the plasmid origin of replication. However, in the homoplasmid state the deletions affected neither copy number nor plasmid stability. The Cmp phenotype is orientation independent, and cmp defects could not be complemented in trans.     

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.     

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.     

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.     

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.     

Modification of the plasmid initiator protein RepC active site during replication

Abstract pT181 is a Staphylococcus aureus rolling circle replicating plasmid whose copy number is controlled by regulating the synthesis and activity of the initiator protein, RepC. The RepC dimer is modified during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. To purify RepC*, RepC was expressed in S. aureus as a fusion protein with a polyhistidine tail. The histidine-tagged RepC retains its initiation and topoisomerase activities in vitro. Histagged RepC/RepC and RepC/RepC* were purified in a two-step procedure. Peptide mapping, mass spectrometric analysis and protein sequencing of purified RepC and RepC* were carried out, and both proteins appeared identical, except that the peptide containing the RepC active site tyrosine used in nicking activity was absent when the purified RepC* sample was analyzed. The absence of the active site in RepC* suggests that this site was modified during replication. The results provide the first direct biochemical evidence that RepC* is a modified form of RepC, and support a model in which RepC replication of pT181 leaves RepC with an oligonucleotide blocking the active site of one of its subunits.     

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.     

Importance of the C terminus of plasmid Rts1 RepA protein for replication and incompatibility of the plasmid.

RepA protein, essential for replication of plasmid Rts1, was found to bind in vivo immediately upstream of the repA promoter in studies with mini-Rts1 derivatives with deletions in the upstream region of repA. We constructed another series of repA mutants that would encode RepA derivatives containing oligopeptide substitutions in place of the carboxyl-terminal six amino acids. These modified RepA proteins could not activate ori (Rts1) at all and showed various degrees of incompatibility, or no incompatibility, toward a mini-Rts1 plasmid. These results suggest that the carboxyl-terminal six (or fewer) amino acids of RepA are important for exerting replication and incompatibility functions. One of the RepA derivatives, which showed an evident incompatibility without initiating replication, was examined for its ability to repress the repA gene.     

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.     

Conformation of a plasmid replication initiator protein affects its proteolysis by ClpXP system

Proteins from the Rep family of DNA replication initiators exist mainly as dimers, but only monomers can initiate DNA replication by interaction with the replication origin (ori). In this study, we investigated both the activation (monomerization) and the degradation of the broad‐host‐range plasmid RK2 replication initiation protein TrfA, which we found to be a member of a class of DNA replication initiators containing winged helix (WH) domains. Our in vivo and in vitro experiments demonstrated that the ClpX‐dependent activation of TrfA leading to replicationally active protein monomers and mutations affecting TrfA dimer formation, result in the inhibition of TrfA protein degradation by the ClpXP proteolytic system. These data revealed that the TrfA monomers and dimers are degraded at substantially different rates. Our data also show that the plasmid replication initiator activity and stability in E. coli cells are affected by ClpXP system only when the protein sustains dimeric form.     

Open-complex formation by the host initiator, DnaA, at the origin of P1 plasmid replication.

Replication of P1 plasmid requires both the plasmid-specific initiator, RepA, and the host initiator, DnaA. Here we show that DnaA can make the P1 origin reactive to the single-strand specific reagents KMnO4 and mung bean nuclease. Addition of RepA further increased the KMnO4 reactivity of the origin, although RepA alone did not influence the reaction. The increased reactivity implies that the two initiators interact in some way to alter the origin conformation. The KMnO4 reactivity was restricted to one strand of the origin. We suggest that the roles of DnaA in P1 plasmid and bacterial replication are similar: origin opening and loading of the DnaB helicase. The strand-bias in chemical reactivity at the P1 origin most likely indicates that only one of the strands is used for the loading of DnaB, a scenario consistent with the unidirectional replication of the plasmid.     

Plasmid copy number mutation in repA gene encoding RepA replication initiator of cryptic plasmid pHM1519 in Corynebacterium glutamicum

ABSTRACT

Cryptic plasmid pHM1519 is a rolling-circular replication mode plasmid of the pCG1 plasmid family in coryneform bacteria. The derived shuttle vector pPK4 is maintained at about 40–50 copies per chromosome in Corynebacterium glutamicum 2256 (ATCC 13869). We found that a mutation (designated copA1) within the repA gene encoding essential initiator protein RepA of the pHM1519-replicon increased the copy number of the mutant plasmid to about 800 copies per chromosome. The mutation was a single G to A base transition, which changed Gly to Glu at position 429 of the amino acid sequence of RepA. In silico secondary structure prediction of RepA suggested that Gly429 is situated in a disordered region in a helix-turn-helix motif, which is a typical DNA-binding domain. This study shows the first example of a high copy number of a C. glutamicum cryptic plasmid caused by an altered replication initiator protein.     

Fluorescence studies of the replication initiator protein RepA in complex with operator and iteron sequences and free in solution

RepA, the replication initiator protein from the Pseudomonas plasmid pPS10, regulates plasmid replication and copy number. It is capable of autorepression, in which case it binds as a dimer to the inverted repeat operator sequence preceding its own gene. RepA initiates plasmid replication by binding as a monomer to a series of four adjacent iterons, which contain the same half-repeat as found in the operator sequence. RepA contains two domains, one of which binds specifically to the half-repeat. The other is the dimerization domain, which is involved in protein-protein interactions in the dimeric RepA-operon complex, but which actually binds DNA in the monomeric RepA-iteron complex. Here, detailed fluorescence studies on RepA and an N-(iodoacetyl)aminoethyl-8-naphthylamine-1-sulfonic acid-labeled single-cysteine mutant of RepA (Cys160) are described. Using time-resolved fluorescence depolarization measurements, the global rotational correlation times of RepA free in solution and bound to the operator and to two distinct iteron dsDNA oligonucleotides were determined. These provide indications that, in addition to the monomeric RepA-iteron complex, a stable dimeric RepA-iteron complex can also exist. Further, F?rster resonance energy transfer between Trp94, located in the dimerization domain, and N-(iodoacetyl)aminoethyl-8-naphthylamine-1-sulfonic acid-Cys160, located on the DNA-binding domain, is observed and used to estimate the distance between the two fluorophores. This distance may serve as an indicator of the orientation between both domains in the unbound protein and RepA bound to the various cognate DNA sequences. No major change in distance is observed and this is taken as evidence for little to no re-orientation of both domains upon complex formation.     

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.     

Interactions between the RepB initiator protein of plasmid pMV158 and two distant DNA regions within the origin of replication

Plasmids replicating by the rolling circle mode usually possess a single site for binding of the initiator protein at the origin of replication. The origin of pMV158 is different in that it possesses two distant binding regions for the initiator RepB. One region was located close to the site where RepB introduces the replication-initiating nick, within the nic locus; the other, the bind locus, is 84bp downstream from the nick site. Binding of RepB to the bind locus was of higher affinity and stability than to the nic locus. Contacts of RepB with the bind and nic loci were determined through high-resolution footprinting. Upon binding of RepB, the DNA of the bind locus follows a winding path in its contact with the protein, resulting in local distortion and bending of the double-helix. On supercoiled DNA, simultaneous interaction of RepB with both loci favoured extrusion of the hairpin structure harbouring the nick site while causing a strong DNA distortion around the bind locus. This suggests interplay between the two RepB binding sites, which could facilitate loading of the initiator protein to the nic locus and the acquisition of the appropriate configuration of the supercoiled DNA substrate.