P1 Plasmid Research Articles

Information Analysis of Sequences that Bind the Replication Initiator RepA

The replication initiator protein RepA of plasmid P1 can bind to 14 sites on the plasmid. These sites are variously used to autoregulate RepA synthesis and for initiation and control of DNA replication. Analysis of information (degree of conservation) at the sites revealed three sequence patches of high conservation. By saturation mutagenesis, the conservation at the outer two patches was found to contribute to RepA binding more critically. The guanine bases that are likely to contact RepA through the major groove were identified by methylation interference and methylation protection experiments. These bases mapped to the outer two patches and were separated by one turn of the helix. Therefore, they belong to major grooves on the same face of DNA. All backbone contacts of the protein, determined by hydroxyl radical footprinting, also mapped to the same face. We conclude from this that RepA binds to its site on one face of the DNA.Information analysis of binding sites for several prokaryotic repressors and activators, where the nature of DNA—protein contacts are known, revealed a correlation between the positions of high conservation and the positions of major grooves that faced the protein. The middle patch of high conservation in the RepA binding sites is an exception since in this region a minor groove is likely to face the protein. The simplest model for minor groove contacts suggests that in B-form DNA a T · A base-pair cannot easily be distinguished from an A · T pair by inspection of the minor groove. Yet in the RepA site, a T → A mutation in the middle patch significantly affects binding. Therefore, the simplest models for both minor and major groove contacts are unlikely. It is possible that the patch determines the proper conformation of the site and thereby contributes to recognition indirectly.

Regulatory interactions between RepA, an essential replication protein, and the DNA repeats of RepFIB from plasmid P307.

The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene (BCDD'D" and EFGHIJ). In vivo genetic tests demonstrate that the BCDD'D" repeats form part of the origin of replication, while some of the downstream repeat elements (HIJ) are involved in the sensing and setting of plasmid copy number. RepA DNA binding to these groups of repeats has been investigated in vivo by utilizing the fact that the replicon contains three active promoters (orip, repAp, and EFp), one of which has previously been shown to control the expression of repA (repAp). All three promoters are closely associated with the repeat elements flanking repA, and an investigation using lacZ or cml gene fusions has demonstrated that RepA expressed in trans is able to repress each promoter. However, these assays suggest that the transcriptional responses of orip and repAp to RepA repression are significantly different, despite the fact that both promoters are embedded within the BCDD'D" repeat elements. Extra copies of the BCDD'D" or EFG repeats in trans have no effect on RepA repression of repAp embedded in a second copy of the BCDD'D" repeats, but copies of the HIJ or EFGHIJ repeats are able to derepress repAp, suggesting that there is a fundamental difference between RepA-BCDD'D" or -HIJ complexes and RepA-EFG or -EFGHIJ complexes.

Activation of DNA Binding by the Monomeric Form of the P1 Replication Initiator RepA by Heat Shock Proteins DnaJ and DnaK

RepA protein of plasmid P1 binds to arrays of 19 bp repeat sequences (iterons) and mediates initiation of replication and its control. Escherichia coli heat shock proteins DnaJ and DnaK can stimulate iteron binding activity of RepA in an ATP-dependent fashion. It has been proposed that RepA binds to DNA as monomers and that the stimulation in binding involves monomerization of RepA dimers which are inactive in the binding reaction. RepA-iteron and RepA-RepA interactions have been measured in this study to determine the equilibrium constants of the two reactions. The apparent K D value for RepA-iteron binding decreased from 10 nM to no more than 0·2 nM at increasing concentrations of the heat shock proteins. The stimulation of binding appears to be due to an increase in active RepA fraction and not to a change in the maximum binding capacity of the active species. This view was deduced from measurements of active RepA fraction, which increased in the presence of heat shock proteins, and from measurements of dissociation rate constants, which were independent of the heat shock protein concentrations. Accounting for the active fractions, the true K D value was estimated to be 0·10(±0·09) nM in 20 mM Tris·HCl (pH 8), 100 mM NaCl, 40 mM KCl, 10 mM MgCl 2, 1 mM dithiothreitol, 0·1 mM EDTA, ATP (50 μM), bovine serum albumin (50 μg/ml), calf thymus DNA (50 μg/ml) and glycerol (5%). The dissociation rate constant was 1·5 × 10 -2 s -1 and the calculated association rate constant was 1·5 × 10 8 M -1 s -1. Ultracentrifugation analyses of RepA at 15,000 r.p.m. in the above buffer but without ATP, bovine serum albumin, calf thymus DNA and glycerol, revealed that the protein was in monomer-direct equilibrium with a K D of 2·6(±0.2) μM at 5°C. Therefore, at protein concentrations used in the binding reactions, RepA is monomeric (>99·5%), in confirmation of the earlier result that RepA binds as a monomer. It follows that the species that is stimulated to bind by the heat shock proteins is also a monomeric form of RepA.

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.

Conformation of the Origin of P1 Plasmid Replication: Initiator Protein Induced Wrapping and Intrinsic Unstacking

The origin of plasmid DNA replication in bacteriophage P1 has five 19 base-pair sites that bind the plasmid-encoded initiator, RepA. Here we show, using a DNA band retardation assay, that RepA can bend DNA that carries one or more of the RepA binding sites. RepA binding to supercoiled DNA carrying the five sites, directly repeated and phased two turns of B-DNA apart, absorbs about one positive superhelical turn of DNA as determined by two-dimensional gel electrophoresis. This indicates that the DNA is wrapped around RepA as a consequence of in-phase bending at the individual binding sites. The RepA-DNA complexes did not show elevated sensitivity to KMnO 4, a reagent specific for pyrimidine bases (T » C) in unstacked DNA. The wrapping of the DNA around RepA, therefore, does not lead to significant unwinding of the double helix. Extensive unwinding, suitable for the initiation of DNA replication, most likely requires participation of factors other than RepA. We also noted that the thymine bases of the sequence 5′-ATC-3′, of which there are 20 in the origin, all reacted to KMnO4 strongly whether or not RepA was present. The preferential reactivity of ATC sequences was specific to the origin region, as thymine residues including those in the ATC sequences did not display elevated sensitivity to KMnO 4 in a DNA fragment from pBR322. On one of two strands of the origin the selective reactivity at the ATC sequences was supercoiling dependent. These results indicate that the origin includes unstacked DNA bases, the significance of which remains to be determined.

The P1 plasmid partition complex at parS. II. Analysis of ParB protein binding activity and specificity.

The P1 plasmid prophage is partitioned by a very high affinity protein complex at its partition site, parS, that contains the P1 ParB protein and Escherichia coli integration host factor (IHF). ParB binds to regions of parS that flank the IHF binding site. In this report, we have examined the sequences to which ParB binds, the spatial relationship between them, and the effect of IHF on ParB binding patterns. Methylation protection and interference experiments were performed on supercoiled plasmids. Mutations that interfered with the action of both proteins in vivo were identified following random mutagenesis of parS. These studies revealed that ParB binds to a complicated, nonsymmetrical region in the right side of parS. ParB recognizes a partial copy of this sequence, TCGCCA, in the left side of parS with much lower affinity. The presence of IHF greatly facilitates the interaction of ParB with parS such that both sides bind with an equal affinity that is much greater than to either side alone. The stimulation by IHF is strongly influenced by helical phasing. These observations support the proposal that ParB is directed, by the bend created by IHF, to bind simultaneously to properly placed sequences flanking the IHF site.

Iterons of stringently controlled plasmids and DNA fingerprinting.

DNA probes which detect polymorphic, repetitive sequences in a variety of genomes have been developed using different approaches. Naturally occurring plasmids, with repeated units termed iterons near or within their origins (ori) or replication, could be of interest for the development of probes, possibly even revealing novel minisatellite families in mammals involved in replicational processes. We used the plasmids P1, pSC101 and RSF1010 or their PCR amplified ori regions as probes in Southern blot hybridisations with mammalian DNA. At low stringency they generated reproducible fingerprint-like patterns. A bovine genomic library was screened at the same stringency with the PCR-amplified ori region of P1 containing the five times repeated core sequence 5'-ATGTGTGNTGNNGGG-3' to generate a probe for cattle DNA with higher specificity.

Expression and regulation of the RepA protein of the RepFIB replicon from plasmid P307.

The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene. Limited N-terminal sequence information obtained from a RepA:beta-galactosidase fusion protein indicates that although the first residue of RepA is methionine, the initiation of translation of RepA occurs from a CTG codon rather than from the predicted GTG codon located further downstream. Overexpressed RepA in trans is capable of repressing a repA:lacZ fusion plasmid in which the expression of the fusion protein is under the control of the repA promoter. The repA promoter has been located functionally by testing a series of repA:lacZ fusion plasmids. Both in vivo genetic tests and in vitro DNA-binding studies indicate that repA autoregulation can be achieved by RepA binding to one or more repeat elements which overlap the repA promoter sequence.

Function of the N-terminal half of RepA in activation of Rts1 ori.

The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for replication. A mutant repA gene, repA delta C143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but at a low frequency, an Escherichia coli polA strain, JG112, when repA delta C143 was cloned into pBR322 with Rts1 ori in the natural configuration. The transformation was less efficient without the dyad DnaA box in the ori region, and no transformation occurred at 42 degrees C, characteristic of Rts1 replication. A fusion of the 3'-terminal half of repA of the P1 plasmid to repA delta C143 yielded a pBR322 chimeric plasmid that contained Rts1 ori through hybrid (Rts1-P1) repA. This plasmid was maintained much more stably in JG112 at 37 degrees C. At 42 degrees C, however, it was quite unstable. The overproduced hybrid RepA protein showed interference with mini-Rts1 replication in trans and also exhibited an autorepressor function, although both activities were decreased. These findings suggest that the N-terminal half of the RepA molecule of Rts1 is involved in the activation of the replication origin.

DnaJ, DnaK, and GrpE heat shock proteins are required in oriP1 DNA replication solely at the RepA monomerization step.

We have found that three Escherichia coli heat shock proteins, DnaK (the hsp70 homolog), DnaJ, and GrpE, function in oriP1 DNA replication in vitro solely to activate DNA binding by the replication initiator protein RepA. Activation results from the conversion of P1 or P7 RepA dimers to monomers that bind with high affinity to the origin of replication of plasmid P1. Thus, the essential role of these three heat shock proteins in this replication system is to change the quaternary structure of a single protein, RepA.

Biochemical activities of the parA partition protein of the P1 plasmid.

The unit-copy P1 plasmid depends for stability on a plasmid-encoded partition region called par, consisting of the parA and parB genes and the parS site. ParA is absolutely required for partition, but its partition-critical role is not known. Purified ParA protein is shown to possess an ATPase activity in vitro which is specifically stimulated by purified ParB protein and by DNA. ParA is responsible for regulation of expression of parA and parB, and purified ParA has an ATP-dependent, site-specific DNA binding activity which recognizes a sequence that overlaps the parA promoter. The role of the ATP-dependence of the binding activity, as well as other possible functions of the ATPase activity in partition, is discussed.

A phage T4 in vitro packaging system for cloning long DNA molecules

Recombinant plasmid DNAs containing long DNA inserts that can be propagated in Escherichia coli would be useful in the analysis of complex genomes. We tested a bacteriophage T4 in vitro DNA packaging system that has the capacity to package about 170 kb of DNA into its capsid for cloning long DNA fragments. We first asked whether the T4 in vitro system can package foreign DNA such as concatemerized λ imm434 DNA and phage P1-pBR322 hybrid DNA. The data suggest that the T4 system can package foreign DNA as efficiently as the mature phage T4 DNA. We then tested the system for its ability to clone foreign DNA fragments using the P1-pBR322 hybrid vectors constructed by Sternberg [Proc. Natl. Acad. Sci. USA 87 (1990) 103–107]. E. coli genomic DNA fragments were ligated with the P1 vectors containing two directly oriented loxP sites, and the ligated DNA was packaged by the T4 in vitro system. The packaged DNA was then transduced into E. coli expressing the phage P1 cyclization recombination protein recombinase to circularize the DNA by recombination between the loxP sites situated at the ends of the transduced DNA molecule. Clones with long DNA inserts were obtained by using this approach, and these were maintained as single-copy plasmids under the control of the P1 plasmid replicon. Clones with up to about 122-kb size inserts were recovered using this approach.

Antiparallel plasmid-plasmid pairing may control P1 plasmid replication.

The copy number of the P1 plasmid replicon is stringently controlled, giving only one or two copies per newborn cell. Control is achieved by the action of the copy-control locus incA, which contains nine repeats of the 19-basepair binding site for the plasmid-encoded initiator protein RepA. A set of five similar repeats are present in the replication origin where RepA acts to trigger initiation. Using an in vitro replication system consisting of an Escherichia coli extract, the P1 origin as a template, and purified RepA protein, we show that supercoiled DNA circles containing the incA locus block origin function in trans. Shutdown becomes complete at a 1:1 ratio of origin to incA sequences. This is not due to titration of the RepA protein, as an excess of RepA can be added without restoring activity. Rather, the incA sequences appear to block the origin by direct contact in a plasmid-plasmid pairing event. When both the origin and the incA locus are present on one plasmid, trans contacts with daughter molecules appear to predominate over cis looping. The results are consistent with a model for replication control where daughter plasmids block their own replication by a pairing in which each origin is in contact with the incA locus of its partner.

Monomerization of RepA dimers by heat shock proteins activates binding to DNA replication origin.

DnaK is a major heat shock protein of Escherichia coli and the homolog of hsp70 in eukaryotes. We demonstrate the mechanism by which DnaK and another heat shock protein, DnaJ, render the plasmid P1 initiator RepA 100-fold more active for binding to the P1 origin of replication. Activation is the conversion of RepA dimers into monomers in an ATP-dependent reaction and the monomer form binds with high affinity to oriP1 DNA. Reversible chemical denaturants also convert RepA dimers to monomers and simultaneously activate oriP1 DNA binding. Increasing protein concentration converts monomers to dimers and deactivates RepA. Based on our data and previous work, we present a model for heat shock protein action under normal and stress conditions.

The P1 plasmid partition complex at parS. The influence of Escherichia coli integration host factor and of substrate topology

The P1 ParB protein is required for active partition and thus stable inheritance of the plasmid prophage. ParB and the Escherichia coli protein integration host factor (IHF) participate in the assembly of a partition complex at the centromere-like site parS. In this report the role of IHF in the formation of the partition complex has been explored. First, ParB protein was purified for these studies, which revealed that ParB forms a dimer in solution. Next, the IHF binding site was mapped to a 29-base pair region within parS, including the sequence TAACTGACTGTTT (which differs from the IHF consensus in two positions). IHF induced a strong bend in the DNA at its binding site. Versions of parS which have lost or damaged the IHF binding site bound ParB with greatly reduced affinity in vitro and in vivo. Measurements of binding constants showed that IHF increased ParB affinity for the wild-type parS site by about 10,000-fold. Finally, DNA supercoiling improved ParB binding in the presence of IHF but not in its absence. These observations led to the proposal that IHF and superhelicity assist ParB by promoting its precise positioning at parS, a spatial arrangement that results in a high affinity of ParB for parS.

Critical sequences in the core of the P1 plasmid replication origin

The core of the P1 plasmid replication origin consists of a series of 7-bp repeats and a G+C-rich stretch. Methylation of the GATC sequences in the repeats is essential. Forty different single-base mutations in the region were isolated and assayed for origin function. A single-base change within any 7-bp repeat could block the origin, irrespective of whether GATC bases were affected. The repeats themselves were critical, but the short intervals between them were not. Mutations in the G+C-rich region showed it to be a spacer whose exact length is important but whose sequence can vary considerably. It maintains a precise distance between the 7-bp repeats and binding sites for the P1 RepA initiator protein. It may also serve as a clamp to limit strand separation during initiation.

Fine-structure analysis of the P1 plasmid partition site

P1 plasmid partition requires two plasmid-encoded Par proteins and a cis-acting site. The site, parS, lies in a region consisting of a 13-bp palindrome and an adjacent AT-rich sequence. A series of point mutations were analyzed for their effects on partition site activity. The results indicated that only the left arm of the palindrome and some adjacent bases were needed. The limits of the functional site were further refined to a maximum of 22 bp, which includes binding sites for the P1 ParB protein. Mutations in the 22-bp site cause concomitant defects in partition and the ability to exert partition-mediated incompatibility. Like the region immediately to the left of the 22-bp region, the right arm of the palindrome is not essential for partition but does contain information that affects the specificity of incompatibility.

Nucleotide sequence and transcriptional analysis of a mitochondrial plasmid from a cytoplasmic male-sterile line of sunflower

A mitochondrial plasmid of 1,939 bp (P2) from a cytoplasmic male-sterile line of sunflower has been cloned and sequenced. It presents 437 bp of near-perfect homology to the 1.4-kb mitochondrial plasmid P1 from sunflower. Sequences homologous to P2 were found in nuclear DNA. P2 was transcribed into a major 980-nucleotide (nt) RNA molecule and two minor transcripts of 570 and 520 nt. They were all transcribed from the same strand and within the region nonhomologous to P1. A single 5' boundary and three 3' termini were determined for P2 transcripts. The 5' end is similar to a consensus sequence for plant mitochondrial genes. No evidence of translation products can be provided.

Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA

Heat-shock proteins are normal constituents of cells whose synthesis is increased on exposure to various forms of stress. They are interesting because of their ubiquity and high conservation during evolution. Two families of heat-shock proteins, hsp60s and hsp70s, have been implicated in accelerating protein folding and oligomerization and also in maintaining proteins in an unfolded state, thus facilitating membrane transport. The Escherichia coli hsp70 analogue, DnaK, and two other heat-shock proteins, DnaJ and GrpE, are required for cell viability at high temperatures and are involved in DNA replication of phage lambda and plasmids P1 and F. These three proteins are involved in replication in vitro of P1 DNA along with many host replication proteins and the P1 RepA initiator protein. RepA exists in a stable protein complex with DnaJ containing a dimer each of RepA and DnaJ. We report here that DnaK and DnaJ mediate an alteration in the P1 initiator protein, rendering it much more active for oriP1 DNA binding.

Unique sequence requirements for the P1 plasmid replication origin

We have carried out a detailed genetic analysis of the P1 plasmid replication origin and shown that it has four major structural requirements: the DnaA box, a series of five 7-base pair (bp) repeats, a GC-rich spacer and five 19-bp repeats that bind the P1 RepA protein. The origin requires the DnaA protein and its recognition sequence (the DnaA box). However, although give boxes are present in two separate blocks in the wild type, just one, placed either to the left or right of the core origin sequences, is sufficient for function as long as the box conforms exactly to the proposed consensus. Each of the five 7-bp repeats that constitute the core of the origin is required; mutations within any of the first six base pairs can block origin function. The required bases include, but are not limited to, those constituting dam methylation sites. Also essential is a 39-bp GC-rich sequence. We show this to be a spacer of critical length that separates the 7-bp repeats from the last required region; a series of 19-bp repeats that bind the P1 RepA initiator protein.