oriC Plasmid Research Articles

OriC plasmids do not affect timing of chromosome replication in Escherichia coli K12

The variability of the time interval between successive rounds of chromosome replication was estimated by density-shift experiments, by measuring the conversion of heavy DNA to hybrid density and light DNAs upon transfer of a steady-state culture growing in medium with [13C]glucose and 15NH4Cl to medium with light isotopes. The coefficient of variation (CV%) for the interreplication time of the Escherichia coli K12 chromosome was found to be 17%, i.e. similar to that for interdivision time. The presence of additional copies of oriC in the cell on a high copy number plasmid did not increase the CV of interreplication time. It is concluded that a single rate-limiting event is unlikely to time the initiation of chromosome replication. The regulation of initiation at oriC and the coordination with cell division is discussed.

Replication of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage P1 and the dnaA gene of Escherichia coli.

We have developed an in vitro DNA-replication system that replicates exogenously added mini-P1 plasmid DNA. The system consists of purified P1 RepA protein and a partially purified mixture of Escherichia coli replication proteins. It is essentially the same as that described for the replication of oriC plasmid DNA [Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374]. Mini-P1 DNA replication requires the E. coli DnaA initiation protein in addition to the P1 RepA initiation protein. The reaction is inhibited by rifampicin, novobiocin, and antibody to DnaB, suggesting the involvement of RNA polymerase, DNA gyrase, and DnaB protein. Replication is initiated in the region of the P1 origin of replication and proceeds unidirectionally as determined by electron microscopy. Thus, the in vitro system mimics the essential features of mini-P1 replication as suggested by genetic studies.

Enzyme systems initiating replication at the origin of the ESCHERICHIA COLI chromosome

More than ten proteins are known to participate in replication of plasmids bearing the unique origin of the Escherichia coli chromosome (oriC). Initiation of replication of oriC plasmids has been resolved into five separable stages. An initial complex formation (Stage I) requires an oriC plasmid, dnaA protein and HU protein. In the presence of ATP at a temperature of greater than 28 degrees C, a dnaB-C protein complex interacts to form a prepriming complex (Stage II). This is followed by extensive unwinding of the template that depends on the further addition of gyrase and single-strand binding protein (SSB) (Stage III). Hydrolysis of an rNTP by dnaB protein (a helicase action) and of ATP by gyrase (a swivelling action) drives the extreme unwinding of the template. This unwound template-protein complex is the substrate for priming by primase (Stage IV) and elongation by DNA polymerase III holoenzyme (Stage V). Priming of all DNA chains is done by primase; RNA polymerase functions in template activation rather than priming. DNA polymerase III holoenzyme, composed of at least seven subunits, synthesizes the DNA chains. The alpha subunit is the polymerase, the epsilon subunit is the 3'----5' exonuclease; alpha + epsilon is the proofreading activity. Following the synthesis of new DNA chains, DNA polymerase I and ribonuclease H remove the RNA primers, polymerase I fills the gaps, and ligase seals the daughter strands (Stage VI). Replication produces plasmids identical in structure and sequence to the initial template.

Nonrandom minichromosome replication in Escherichia coli K-12

The intervals between rounds of chromosome and minichromosome replication were measured by density shift experiments and found to be similar. Thus the minichromosome, a lambda asnA oriC bacteriophage, mostly replicates once each division cycle rather than randomly, despite its high copy number. Slight differences between the chromosome and the oriC plasmid are explained.

Regulation of transcription of the chromosomal dnaA gene of Escherichia coli.

By comparative S1 analysis we investigated the in vivo regulation of transcription of the chromosomal dnaA gene coding for a protein essential for the initiation of replication at the chromosomal origin. Inactivation of the protein in dnaA mutants results in derepression, whereas excess DnaA protein (presence of a DnaA overproducing plasmid) leads to repression of dnaA transcription. Both dnaA promoters are subject to autoregulation allowing modulation of transcriptional efficiency by at least 20-fold. Increasing the number of oriC sequences (number of DnaA binding sites) in the cell by introducing oriC plasmids leads to a derepression of transcription. Autoregulation and binding to oriC suggest that the DnaA protein exerts a major role in the regulation of the frequency of initiation at oriC. The efficiency of transcription of the dnaA2 promoter is reduced in the absence of dam methylation, which is involved in the regulation of oriC replication.

Asymmetric replication of an oriC plasmid in Escherichia coli.

Plasmid pTSO118 containing the Escherichia coli origin of replication, oriC, initiated replication simultaneously with the chromosome when temperature-sensitive host cells were synchronized by temperature shifts. Replicating intermediates of the plasmid as well as of the chromosome were isolated from the outer membrane fraction of the cell. Plasmid DNA with eye structures was enriched when cytosine-1-beta-arabinofuranoside was introduced into the culture during replication. Electron microscopy of the replicating molecules, after digestion with restriction endonucleases, showed that the replication fork proceeds exclusively counter-clockwise towards the unc operon. We conclude that the replication of the oriC plasmid is unidirectional or, if bidirectional, is highly asymmetric.

Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome.

During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template. Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive. Semi-conservative replication proceeds via delta structure as intermediates. Daughter molecules include nicked intermediates. Daughter molecules include nicked monomers and catenated pairs. Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow. Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonuclease H, gap filling, and ligation of nascent strands by ligase. Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.

Dissection of promoter sequences involved in transcriptional activation of theEscherichia colireplication origin

The replication frequency of oriC plasmids in vivo is positively affected by specific transcripts running into oriC. These transcripts that activate oriC are initiated at a promoter of a gene coding for a 16kD protein. Genetic evidence is presented for binding of the initiation factor dnaA to a specific sequence (dnaA box) upstream of this promoter. Binding of the dnaA protein to this dnaA box regulates transcription initiation negatively. It was also demonstrated that binding of dnaA protein to the 16kD promoter region is essential to accomplish the actual activation event within the origin. Replication and incompatibility experiments suggest that dnaA protein is present within the activating transcription complex. The function of dnaA in this replication control mechanism is discussed.

Negative control of oriC plasmid replication by transcription of the oriC region.

We have demonstrated that the replication of the oriC plasmid, carrying the replication origin of the Escherichia coli chromosome, is inhibited by transcriptional readthrough from an oriC flanking region of the plasmid. This was drawn from an examination of the replication of an oriC plasmid, pKZ4, which bears the lacOP segment at the right-hand side of oriC (the asnA side) in such an orientation that transcription from the lac promoter proceeds towards oriC. Replication of pKZ4 was found to be drastically inhibited by inducing transcription from the lac promoter with IPTG, an inducer of the lactose operon. When trp transcription attenuator termination sequences were inserted near the right-hand end of the oriC region of pKZ4, the replication of the plasmid became considerably insensitive to the inhibitory effect of IPTG. This indicates that the inhibition is due to the frequent leftward transcription, which initiates at the lac promoter and proceeds into the oriC region. Since IPTG inhibits the replication of pKZ4, but not that of another coexisting oriC plasmid which is devoid of the lacOP segment, the replication inhibition is judged to act only in cis. Transcription from the promoter of the chloramphenicol resistance gene also caused the inhibition of oriC plasmid replication.

Initiation of enzymatic replication at the origin of the Escherichia coli chromosome: primase as the sole priming enzyme.

The enzymatic replication of plasmids containing the unique (245 base pair) origin of the Escherichia coli chromosome (oriC) can be initiated with any of three enzyme priming systems: primase alone, RNA polymerase alone, or both combined (Ogawa, T., Baker, T. A., van der Ende, A. & Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, 3562-3566). At certain levels of auxiliary proteins (topoisomerase I, protein HU, and RNase H), the solo primase system is efficient and responsible for priming synthesis of all DNA strands. Replication of oriC plasmids is here separated into four stages: (i) formation of an isolable, prepriming complex requiring oriC, dnaA protein, dnaB protein, dnaC protein, gyrase, single-strand binding protein, and ATP; (ii) formation of a primed template by primase; (iii) rapid, semiconservative replication by DNA polymerase III holoenzyme; and (iv) conversion of nearly completed daughter molecules to larger DNA forms. Optimal initiation of the leading strand of DNA synthesis, over a range of levels of auxiliary proteins, appears to depend on transcriptional activation of the oriC region by RNA polymerase prior to priming by primase.

Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli.

In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis. Transformation frequencies of oriC plasmids into E. coli dam mutants, deficient in the GATC-specific DNA methylase, are greatly reduced compared with parental dam+ cells, particularly for plasmids that must use oriC for initiation. Mutations that suppress the mismatch repair deficiency of dam mutants do not increase these low transformation frequencies, implicating a new function for the Dam methylase. oriC DNA isolated from dam- cells functions 2- to 4-fold less well in the oriC-specific in vitro initiation system when compared with oriC DNA from dam+ cells. This decreased template activity is restored 2- to 3-fold if the DNA from dam- cells is first methylated with purified Dam methylase. Bacterial origin plasmids or M13-oriC chimeric phage DNA, isolated from either base substitution or insertion dam mutants of E. coli, exhibit some sensitivity to digestion by DpnI, a restriction endonuclease specific for methylated GATC sites, showing that these dam mutants retain some Dam methylation activity. Sites of preferred cleavage are found within the oriC region, as well as in the ColE1-type origin.

Replication initiated at the origin ( oriC) of the E. coli chromosome reconstituted with purified enzymes

A crude soluble enzyme system capable of authentic replication of a variety of oriC plasmids has been replaced by purified proteins constituting three functional classes: initiation proteins (RNA polymerase, dnaA protein, gyrase) that recognize the oriC sequence and presumably prime the leading strand of the replication fork; replication proteins (DNA polymerase III holoenzyme, single-strand binding protein, primosomal proteins) that sustain progress of the replication fork; and specificity proteins (topoisomerase I, RNAase H 1 protein HU) that suppress initiation of replication at sequences other than oriC, coated with dnaA protein. Protein HU and unidentified factors in crude enzyme fractions stimulate replication at one or more stages. Replication has been separated temporally and physically into successive stages of RNA synthesis and DNA synthesis.

Topoisomerase I confers specificity in enzymatic replication of the Escherichia coli chromosomal origin.

Crude soluble enzyme fractions that initiate bidirectional replication from the unique Escherichia coli chromosomal origin (oriC) have been fractionated further to identify the components and mechanisms of this complex system. Among the necessary factors is a class of specificity proteins that suppress initiations on plasmids which lack the oriC sequence and which do not depend on dnaA protein. One such specificity factor has been identified as RNase H (Ogawa, T., Pickett, G. G., Kogoma, T., and Kornberg, A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1040-1044). Another, described here, has proved to be topoisomerase I. A protein was purified to near homogeneity based on assays of (i) inhibition of the replication of plasmids (and other supercoiled DNA) lacking oriC and (ii) conferral of dnaA protein dependence on the replication of an oriC plasmid. This specificity protein is indistinguishable from authentic E. coli topoisomerase I by several criteria: (i) molecular weight under denaturing conditions, (ii) relaxation activity on negatively supercoiled DNA, (iii) cleavage pattern of single-stranded DNA, (iv) specificity factor activity, and (v) neutralization of activity by antibody against topoisomerase I. One possible mechanism of the specificity action of topoisomerase I is destabilization of primers for replication except when they are preserved at an oriC sequence bound by dnaA protein and other replication proteins.

In vitro replication of a dam methylated and non-methylated ori-C plasmid

We have examined the replication of a dam methylated and non-methylated ori-C plasmid in an in vitro ori-C dependent replication system. The results show that the non-methylated plasmid is 50% to 80% less efficient in the initiation of DNA synthesis; that the methylation state of the plasmid does not change the site of initiation at ori-C, and that in both cases initiation at this region requires the presence of exogenously furnished dnaA protein.

Mini-F plasmid genes that couple host cell division to plasmid proliferation.

A mechanism for stable maintenance of plasmids, besides the replication and partition mechanisms, has been found to be specified by genes of a mini-F plasmid. An oriC plasmid carrying both a mini-F segment necessary for partition [coordinates 46.4-49.4 kilobase pairs (kb) on the F map] and another segment (42.9-43.6 kb), designated ccd (coupled cell division), is more stably maintained than are oriC plasmids carrying only the partition segment; the stability is comparable to that of the parental mini-F plasmid. When replication of a plasmid carrying ccd is prevented and the plasmid copy number decreases, to as few as one per cell, host cell division is inhibited, but not increase of turbidity or chromosome replication. Appearance of plasmid-free segregants is therefore effectively prevented under such conditions. Experimental results suggest that reduction of the copy number of plasmids carrying the ccd region causes an inhibition of cell division and that the ccd region can be dissected into two functional regions; one (ccdB) inhibits cell division and the other (ccdA) releases the inhibition. The interplay of the ccdA and ccdB genes promotes stable plasmid maintenance by coupling host cell division to plasmid proliferation.

The 245 base-pair oriC sequence of the E. coli chromosome directs bidirectional replication at an adjacent region.

The replication origin of the E. coli K-12 chromosome has been isolated as autonomously replicating molecules(oriC plasmid), and the DNA region essential for replicating function(oriC) has been localized to a sequence of 232-245 base-pairs(bp) by deletion analysis. In this report, the functional role of oriC was analysed by using an in vitro replication system and various OriC+ and OriC- plasmids previously constructed. The results obtained were summarized as follows: (1) The oriC sequence contained information enough to direct bidirectional replication. (2) The actual DNA replication began at a region near, but outside, oriC and progressed bidirectionally. (3) Initiation of DNA synthesis at the specific region required the dnaA-complementing fraction from cells harboring a dnaA-carrying plasmid.

Incompatibility of plasmids containing the replication origin of the Escherichia coli chromosome.

Plasmids containing the replication origin of the Escherichia coli chromosome (oriC plasmids) are unstable in certain recA strains of E. coli. However, they can be maintained more stably in other recA strains. This stable maintenance has allowed us to study the incompatibility properties of oriC plasmids. We have found that two oriC plasmids are incompatible: they cannot be stably coinherited in individual dividing cells. An oriC plasmid is excluded from growing bacteria at a much faster rate in the presence of a hybrid plasmid made from an oriC plasmid and a high-copy-number vector plasmid than in the presence of another oriC plasmid. By inserting various segments around the oriC region into high-copy-number vectors, we have shown that two different regions in the vicinity of the oriC region determine incompatibility. One region, which we named incA, includes the region essential for autonomous replication of the oriC plasmid. The other, incB, is adjacent to incA but is not required for autonomous replication.

Inhibition of DNA replication in vitro by pefloxacin

Pefloxacin (a novel quinolone antibiotic) is demonstrated to be a drug inhibiting DNA replication 10-times more efficiently than oxolinic acid measured either in toluene-treated E. coli or in an in vitro replication system for oriC plasmids [6]. DNA repair synthesis is not inhibited by the drug.

Copy-number mutants of the plasmid carrying the replication origin of the Escherichia coli chromosome: evidence for a control region of replication.

A composite plasmid (pXX11) was constructed by joining of an oriC plasmid (pMCR115) carrying the replication origin (oriC) of the Escherichia coli chromosome and a mini-F plasmid (pSC138) carrying the ampicillin-resistance gene (bla). Plasmid pXX11 can replicate, by using oriC, in Hfr cells and mafA mutant cells that cannot support replication of an F plasmid. This plasmid is stably maintained in these host cells during cell growth even under nonselective conditions by use of the partition mechanism of the mini-F genome. In contrast to other oriC plasmids reported previously, pXX11 has no detectable effect on host cell growth. Higher copy-number (Cop-) mutants of pXX11 were isolated, and some of them were found to carry an insertion or deletion within a region derived from the E. coli chromosome. This region, designated cop (copy number), covers about 0.7 kilobase pair and is located approximately 3 kilobase pairs away from the oriC region at the side opposite the asn gene. Evidence suggests that the normal cop region locted on the oriC plasmid acts to reduce the copy number of the plasmid. Plasmid pXX11 complements the uncB402 mutation located on the host chromosome, but some of the Cop- plasmids do not, suggesting that the cop region is vey closely linked to uncB.

Cloning of replication origins from the E. coli K12 chromosome

MINIPLASMIDS, containing only an origin of replication and plasmid-coded functions necessary for plasmid replication and maintenance have been isolated from larger plasmids1. The technique used was to cleave the parent plasmid with the restriction endonuclease EcoRI and reanneal and ligate the fragments so generated in the presence of another EcoRI-generated fragment of DNA coding for a β-lactamase. This second fragment was not capable of self-replication but hybrids between it and a plasmid fragment which was able to replicate could be detected because calls of Escherichia coli transformed with such hybrids would be resistant to ampicillin. We have used the same technique to isolate minichromosomes from the total chromosomal DNA of E. coli K12 and have found that two different chromosomal EcoRI fragments can function as autonomously replicating plasmids. One type of plasmid is indistinguishable from that isolated independently by Yasuda and Hirota2 and contains the origin of replication normally active in chromosome replication termed oriC (refs 3–6). The other type, which we call oriJ plasmids to distinguish them from oriC plasmids, are described below. Both types can be isolated from a single strain of E. coli and they contain chromosomal DNA which is not homologous in DNA · DNA hybridisation studies. Consequently we conclude that the E. coli K12 chromosome contains at least two segments of DNA capable of self-replication.