Location of the origin of replication for the 7.5-kb Chlamydia trachomatis plasmid.

The hypothetical origin of replication for the 7.5-kb plasmid common to Chlamydia trachomatis is believed to be in a region of the plasmid that contains four 22-bp tandem repeats preceded by an A-T-rich region. To test this hypothesis, replication of plasmid DNA in metabolically active reticulate bodies of the Lymphogranuloma venereum biovar of C. trachomatis was examined by electron microscopy. The results presented show that the origin of replication appears to be near the tandem repeats of pCHL2. In addition, replication of the 7.5-kb plasmid is unidirectional, and the copy number during replication is 7-10. The evidence presented suggests that C. trachomatis has a homologue to the Escherichia coli dnaA gene and that this homologue might be involved in replication of the C. trachomatis 7.5-kb plasmid.     

Identification of the origin of replication of the eukaryoteDictyostelium discoideum nuclear plasmid Ddp2

Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryoteDictyostelium discoideum. We have identified two functional domains, a large open reading frame (Rep gene) and a 626-bp fragment containing an origin of replication (ori). The ori, when cloned into a shuttle vector, confers stable extrachromosomal replication inD. discoideum, provided that the Rep gene, which acts intrans, is integrated into the host genome. Ddp2 carries a 501-bp imperfect inverted repeat, and part of the ori overlaps with one of these repeats. The ori sequence contains two direct repeats of 49 bp comprising two 10-bp “TGTCATGACA” palindromes separated by a poly(T · A) sequence. Deletion of either 49-bp repeat abolished extrachromosomal replication.     

Recombination between an Flac and a mini-FKm plasmid deleted for an origin of replication

The mini-F plasmid specifying resistance to kanamycin (Km), pML31, contains an origin of replication at kilobase coordinate 42.6 in the F DNA sequences. In previous research we found that this origin could be deleted by recombinant DNA techniques without the loss of plasmid maintenance functions. In this report we show that the deleted plasmid, designated pMF21, has normal incompatibility properties and a recA⁺-dependent ability to form cointegrates with an Flac plasmid. By comparison, pML31 does not form cointegrates with the Flac plasmid at a detectable frequency. The frequency for spontaneous loss of the Lac⁺ phenotype in strains containing pMF21:Flac cointegrates resembles that of the Flac plasmid; however, in some Lac⁻ variants the Km^r phenotype is retained. Examination of the plasmid DNA in four of these Lac⁻Km^r clones revealed two with normal pMF21 plasmids and two with plasmids intermediate in size between pMF21 and the Flac.     

Functional organization of the plasmid pT181 replication origin

Replication of the staphylococcal plasmid pT181 is initiated at the origin (ori) with the introduction of a site-specific nick by the plasmid-encoded initiator protein RepC. Deletion analysis showed that a sequence of about 70 base-pairs is required for full ori function, including the ability to compete with a co-resident wild-type origin for the trans-acting RepC protein. A shorter sequence of 43 base-pairs is sufficient for origin function in the absence of competition. Single and double point mutations within these 43 base-pairs were used to determine the sequence requirement for replication within the minimal origin. Deletion mutants and point mutants were tested in replication and competition assays in vivo and in vitro, and in a RepC-mediated nicking assay.     

The large endogenous plasmid of Anacystis nidulans: Mapping, cloning and localization of the origin of replication

Summary Anacystis nidulans contains two cryptic plasmids of 8.0 (pANS) and 48.5 (pANL) kilobasepairs (kbp). A clone bank of the large plasmid pANL consisting of 7 Bam HI fragments has been established. The cloned fragments were used as radioactive probes to Bam HI, Sal I, Hind III and Eco R1 digests of pANL in blot hybridization experiments to verify the clones and map the restriction fragments. Further size characterization of the physical map was done by restriction analysis of the cloned fragments. The origin of replication has been located in the largest Bam HI fragment of the large plasmid.     

In vivo definition of the functional origin of replication (ori(+ )) of the promiscuous plasmid pLS1

The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein. The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins. Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline. The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex. Sequence analysis of the NocR binding site indicated the presence immediately downstream of the –10 sequence of the nocR promoter of a 12 by putative operator overlapping a consensus gyrase recognition sequence and an 18 by long alternating purine-pyrimidine sequence. These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.     

Additional genes essential for replication of the mini-F plasmid from origin I

Summary We isolated a series of Tn5-insertional mutants from the mini-F plasmid, which has a deletion in the origin II region and replicates exclusively from origin I, and found that the mutants that had Tn5 in either the F4 or the F5 gene were defective in their replication. It is concluded that, in addition to the F3 gene on which we have reported previously, both the F4 and the F5 genes are essential for the replication from origin I.     

An essential gene for replication of the mini-F plasmid from origin I

Summary We constructed a series of defective mini-F plasmids, which have deletion(s) in the replication origin I and/or origin II, and their derivatives, which do not produce F3 protein, by insertion of the XhoI fragment of Tn5 into the XhoI site at 41.0 F (kilobases on the coordinate map of F-plasmid). Using these mutant mini-F plasmids, we found that F3 protein is essential for the replication of mini-F from origin I, but not from origin II.     

Localization of the replication origin of plasmid pE194.

The pE194 replication origin was localized to a 265-base-pair interval by analyzing the ability of purified pE194 restriction fragments to direct replication of heterologous plasmids. Replication was dependent upon RepF protein supplied in trans. The origin region contained a GC-rich dyad symmetry which may serve as the RepF target.     

Identification of the minimal essential region for the replication origin of miniF plasmid

Summary The minimal replication origin of miniF plasmid was found to lie within a region of 217 bp in length. This region contains an AT cluster and the four 19 bp direct repeats responsible for incompatibility, termed incB. Its location coincides with that of ori2 of plasmid F, previously inferred to be the replication starting point by electron microscopic analysis (Eichenlaub et al. 1981).Abbreviations kb kilobase(s) - bp base pairs - Ap ampicillin - Tc tetracycline     

Isolation of the origin of replication of the IncW-group plasmid pSa

The origin of replication of the IncW plasmid pSa has been cloned and the function of this origin in Escherichia coli examined. A 1.9-kb region of DNA is required for efficient autonomous replication, and a 0.47-kb fragment within this region can initiate replication only in the presence of an autonomously replicating derivative of pSa. An Mr 35,000 protein (repA) is encoded adjacent to the origin and is required for efficient initiation of replication. The derivatives examined provide information suggesting a direct role of partition factors in plasmid replication and incompatibility.     

HU protein binding to the replication origin of the rolling-circle plasmid pKYM enhances DNA replication

The RepK protein, which is encoded by the rolling-circle plasmid pKYM, binds to the PR I site in the pKYM DNA replication origin. We have identified HU as a protein that binds to the PR II and PR III sites in the replication-enhancing region which is downstream of PR I. DNA footprinting assays show that HU binds to these two sites only when RepK is bound to PR I, and that HU also enhances the binding of RepK to PR I. In vivo, pKYM was unable to transform an HU null strain. Two mutant RepK proteins, RepKW179Y, which contains a Trp-to-Tyr exchange at position 179, and RepKD277L, which contains an Asp-to-Leu mutation at residue 277, initiate DNA replication in vivo in the absence of HU. In vitro, these mutant RepK proteins form more stable complexes with the pKYM origin region than does the wild-type RepK protein. These results indicate that HU plays a role in the formation of a stable RepK-origin complex, which is required for the initiation of pKYM DNA replication. Received: 24 July 1996 / Accepted: 30 December 1996     

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.     

F plasmid replication and the division cycle of Escherichia coli B/r

A terminal stage in the duplication of many bacterial plasmids involves the transient formation of catenated molecules containing two interlocked monomeric plasmid units. This property of plasmid replication was exploited to examine the relationship between F replication and the division cycle of Escherichia coli B/r cells growing in undisturbed, exponential-phase cultures. Various cultures of F′lac- or FKm^r-containing cells were briefly exposed to [³H]thymidine, and then the transfer of radioactivity into, and out of, a catenated dimer consisting of two closed circular monomers was measured during a chase period. The fraction of plasmid molecules present in this dimer form was determined by separating cellular DNA in alkaline sucrose gradients. In addition, plasmid replication was studied in synchronously growing cultures by measuring both [³H]thymidine incorporation into covalently closed circular DNA and β-galactosidase inducibility. The results suggest that replication of F plasmids can take place throughout the cell division cycle, with the probability of replication increasing toward the end of the cycle. The presence of DNA homologous to the chromosome on the F′lac did not alter the replication pattern of the plasmid during the division cycle.     

Anatomy of the replication origin of plasmid ColE2-P9

The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.     

The in vivo replication origin of the yeast 2 microns plasmid

We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.     

Supercoiling unwinds two-micrometer plasmid yeast DNA at the origin of replication

All studied origins of replication of DNA in Saccharomyces cerevisiae contain DNA unwinding elements. The introduction of unrestrained negative supercoiling leads to melting of the two DNA strands in DNA unwinding elements. To understand the mechanism of DNA replication it is important to know whether the most unstable region of DNA coincides with the origin of replication. Two-micrometer plasmid DNA from S. cerevisiae inserted in pBR322 was investigated by cleaving with snake venom phosphodiesterase. Its single-strand endonucleolytic activity allows cutting of negatively supercoiled DNA in the DNA unwinding elements. The sites of the venom phosphodiesterase hydrolysis were mapped by restriction enzymes. This study shows that the unwinding of the two-micrometers plasmid DNA of S. cerevisiae takes place only in the origin of replication as a result of unrestrained negative supercoiling.