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The regulation of initiation of DNA replication is crucial to ensure that the genome is replicated only once per cell cycle. In the Gram-positive bacterium Bacillus subtilis, the function of the YabA protein in initiation control was assigned based on its interaction with the DnaA initiator and the DnaN sliding clamp in the yeast two-hybrid and on the overinitiation phenotype observed in a yabA null strain. However, YabA is unrelated to known regulators of initiation and interacts with several additional proteins that could also be involved directly or not in initiation control. Here, we investigated the specific role of YabA interactions with DnaA and DnaN in initiation control by identifying single amino acid changes in YabA that disrupted solely the interaction with DnaA or DnaN. These disruptive mutations delineated specific interacting surfaces involving a Zn2+-cluster structure in YabA. In B. subtilis, these YabA interaction mutations abolished both initiation control and the formation of YabA foci at the replication factory. Upon coexpression of deficient YabA mutants, mixed oligomers formed foci at the replisome and restored initiation control, indicating that YabA acts within a heterocomplex with DnaA and DnaN. In agreement, purified YabA oligomerized and formed complexes with DnaA and DnaN. These findings underscore the functional association of YabA with the replication machinery, indicating that YabA regulates initiation through coupling with the elongation of replication.

DNA replication in bacteria is carried out by a multiprotein complex, which is thought to contain only one essential DNA polymerase, specified by the dnaE gene in Escherichia coli and the polC gene in Bacillus subtilis. Bacillus subtilis genome analysis has revealed another DNA polymerase gene, dnaE(BS), which is homologous to dnaE. We show that, in B. subtilis, dnaE(BS) is essential for cell viability and for the elongation step of DNA replication, as is polC, and we conclude that there are two different essential DNA polymerases at the replication fork of B. subtilis, as was previously observed in eukaryotes. dnaE(BS) appears to be involved in the synthesis of the lagging DNA strand and to be associated with the replication factory, which suggests that two different polymerases carry out synthesis of the two DNA strands in B. subtilis and in many other bacteria that contain both polC and dnaE genes.

Phenotypes of Bacillus subtilis priA mutants suggest that they are deficient in the restart of stalled chromosomal replication forks. The presumed activity of PriA in the restart process is to promote the assembly of a multiprotein complex, the primosome, which functions to recruit the replication fork helicase onto the DNA. We have proposed previously that three proteins involved in the initiation of replication at oriC in B. subtilis, DnaB, DnaD and DnaI, are components of the PriA primosome in this bacterium. However, the involvement of these proteins in replication restart has not yet been studied. Here, we describe dnaB mutations that suppress the phenotypes of B. subtilis priA mutants. In a representative mutant, the DnaC helicase is loaded onto single-stranded DNA in a PriA-independent, DnaD- and DnaI-dependent manner. These observations confirm that DnaB, DnaD and DnaI are primosomal proteins in B. subtilis. Moreover, their involvement in the suppression of priA phenotypes shows that they participate in replication fork restart in B. subtilis.

Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base., (Copyright 2001 Academic Press.)

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process.

We have studied DNA recombination between 513 bp tandem direct repeats present in a kanamycin resistance gene inserted in the Bacillus subtilis chromosome. Tandem repeat deletion was not significantly affected by a recA mutation. However, recombination was stimulated by mutations in genes encoding replication proteins, including the primosomal proteins DnaB, DnaD and the DnaG primase, the putative DNA polymerase III subunits PolC, DnaN and DnaX, as well as the DNA polymerase DnaE. Hyper-recombination was found to be dependent on RecA in the dnaE, dnaN and dnaX mutants, whereas the dnaG and dnaD mutants stimulated recombination independently of RecA. Altogether, these data show that both RecA-dependent and RecA-independent mechanisms contribute to recombination between tandem repeats in B. subtilis and that both types of recombination are stimulated by replication mutations.

The holD gene codes for the psi subunit of the Escherichia coli DNA polymerase III holoenzyme, a component of the gamma complex clamp loader. A holD mutant was isolated for the first time in a screen for mutations that increase the frequency of tandem repeat deletions. In contrast to tandem repeat deletions in wild-type strains, deletion events stimulated by the holD mutation require RecA. They do not require RecF, and hence do not result from the recombinational repair of gaps, arguing against uncoupling of the leading and lagging strand polymerases in the holD mutant. The holD recBC combination of mutations is lethal and holD recBts recCts strains suffer DNA double-strand breaks (DSBs) at restrictive temperature. DSBs require the presence of the Holliday junction-specific enzymes RuvABC and are prevented in the presence of RecBCD. We propose that impairment of replication due to the holD mutation causes the arrest of the entire replisome; consequently, Holliday junctions are formed by replication fork reversal, and unequal crossing over during RecA- and RecBCD-mediated re-incorporation of reversed forks causes the hyper-recombination phenotype.

The Escherichia coli UvrD helicase (or helicase II) is known for its involvement in DNA repair. We report that UvrD is required for DNA replication of several different rolling-circle plasmids in E. coli, whereas its homologue, the Rep helicase, is not. Lack of UvrD helicase does not impair the first step of plasmid replication, nicking of the double-stranded origin by the plasmid initiator protein. However, replication proceeds no further without UvrD. Indeed, the nicked plasmid molecules accumulate to a high level in uvrD mutants. We conclude that UvrD is the replicative helicase of various rolling-circle plasmids. This is the first description of a direct implication of UvrD in DNA replication in vivo.

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from Phi29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.

Replication arrest leads to the occurrence of DNA double-stranded breaks (DSB). We studied the mechanism of DSB formation by direct measure of the amount of in vivo linear DNA in Escherichia coli cells that lack the RecBCD recombination complex and by genetic means. The RuvABC proteins, which catalyze migration and cleavage of Holliday junctions, are responsible for the occurrence of DSBs at arrested replication forks. In cells proficient for RecBC, RuvAB is uncoupled from RuvC and DSBs may be prevented. This may be explained if a Holliday junction forms upon replication fork arrest, by annealing of the two nascent strands. RecBCD may act on the double-stranded tail prior to the cleavage of the RuvAB-bound junction by RuvC to rescue the blocked replication fork without breakage.

pAMbeta1 is a plasmid isolated from Enterococcus faecalis which replicates in Bacillus subtilis by a unidirectional theta mechanism. It has been shown previously that initiation of pAMbeta1 replication requires a plasmid-encoded protein (RepE) and a short origin and is carried out by the host DNA polymerase I. It is not known which primer is used by this polymerase for initiating replication. Here, we report that a transcription fork passing through the origin is a limiting factor for plasmid replication. Transcription that activates the origin is initiated at the repE promoter and is thus regulated by the plasmid copy-number control system. Two lines of evidence suggest that the transcription generates the primer for the DNA polymerase I. First, the transcription must start upstream from the origin and progress in the direction of replication to be effective. Second, 3' ends of RNA transcripts initiated upstream of the origin map within the origin, provided that the Rep protein and an intact origin are present. This is the first report for simultaneous requirement of a transcription fork, a replication protein and the DNA polymerase I in initiation of DNA replication.

The mechanisms ensuring regulation of DNA replication in genomes containing multiple replicons are poorly understood. In this report, we addressed this question by analysing in Bacillus subtilis the replication of a derivative of the promiscuous plasmid pIP501 that carries a rolling-circle and a theta replicon. Genetic analyses revealed that the rolling-circle replicon is strongly inhibited in the derivative and that inhibition requires three elements involved in theta replication: the replication origin, the initiator RepR protein and strong transcription of the repR gene. Inhibition is, however, independent of DNA synthesis at the theta origin. We conclude that rolling-circle inhibition is caused by an inhibitory signal encoded by the theta replicon and propose that the signal is composed, at least, of the RepR protein bound to its cognate origin.

The only DNA helicase essential for Escherichia coli viability is DnaB, the chromosome replication for helicase. In contrast, in Bacillus subtilis, in addition to the DnaB counterpart called DnaC, we have found a second essential DNA helicase, called PcrA. It is 40% identical to the Rep and UvrD DNA helicases of E. coli and 61% identical to the PcrA helicase of Staphylococcus aureus. This gene is located at 55 degree on the chromosome and belongs to a putative operon together with a ligase gene (lig) and two unknown genes named pcrB and yerH. As PcrA was essential for cell viability, conditional mutants were constructed. In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling-circle replication of the plasmid pT181 was inhibited. Analysis of the replication intermediates showed that leading-strand synthesis of pT181 was prevented upon PcrA depletion. To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E. coli. PcrA suppressed the UV sensitivity defect at a uvrD mutant but not its mutator phenotype. Furthermore, it conferred a Rep-phenotype on E. coli. Altogether, these results show that PcrA is an helicase used for plasmid rolling-circle replication and suggest that it is also involved in UV repair.

A number of large extrachromosomal elements encode prokaryotic type I topoisomerases of unknown functions. Here, we analysed the topoisomerase Topbeta encoded by the Gram-positive broad-host-range plasmid pAMbeta1. We show that this enzyme possesses the DNA relaxation activity of type I topoisomerases. Interestingly, it is active only on plasmids that use DNA polymerase I to initiate replication, such as pAMbeta1, and depends on the activity of this polymerase. This is the first example, to our knowledge, of prokaryotic type I topoisomerase that is specific for a given type of replicon. During pAMbeta1 replication in Bacillus subtilis cells, Topbeta promotes premature arrest of DNA polymerase I, approximately 190bp downstream of the replication initiation point. We propose that Topbeta acts on the early replication intermediates of pAMbeta1, which contain D-loops formed by DNA polymerase I-mediated strand displacement. The possible role of the resulting DNA Pol I arrest in plasmid replication is discussed.

UV light irradiation increases genetic instability by causing mutations and deletions. The mechanism of UV-induced rearrangements was investigated making use of deletion-prone plasmids. Chimeric plasmids carrying pBR322 and M13 replication origins undergo deletions that join the M13 replication origin to a random nucleotide. A restriction fragment was UV irradiated, introduced into such a hybrid plasmid and deletions formed at the M13 origin were analysed. In most of the deletant molecules, the M13 replication nick site was linked to a nucleotide in the irradiated fragment, showing that UV lesions are deletion hotspots. These deletions were independent of the UvrABC excision repair proteins, suggesting that the deletogenic structure is the lesion itself and not a repair intermediate. They were not found in the absence of M13 replication, indicating that they result from the encounter of the M13 replication fork with the UV lesion. Furthermore, UV-induced deletions occurred independently of pBR322 replication. We conclude that, in contrast to pBR322 replication forks, M13 replication forks blocked by UV lesions are deletion prone. We propose that the deletion-prone properties of a UV-arrested polymerase depend on the associated helicase.

Replication blockage induces non-homologous deletions in Escherichia coli. The mechanism of the formation of these deletions was investigated. A pBR322-mini-oriC hybrid plasmid carrying two E. coli replication terminators (Ter sites) in opposite orientations was used. Deletions which remove at least the pBR322 blocking site (named Ter1) occurred at a frequency of 2 x 10(-6) per generation. They fall into two equally large classes: deletions that join sequences with no homology, and others that join sequences of 3-10 bp of homology. Some 95% of the deletions in the former class resulted from the fusion of sequences immediately preceding the two Ter sites, indicating a direct role for blocked replication forks in their formation. These deletions were not found in a topA10 mutant, suggesting a topoisomerase I-mediated process. In contrast, deletions joining short homologous sequences were not affected by the topA10 mutation. However, the incidence of this second class of deletions increased 10-fold in a recD mutant, devoid of exonuclease V activity. This indicates that linear molecules are intermediates in their formation. In addition, approximately 50% of these deletions were clustered in the region flanking the Ter1 site. We propose that they are produced by repair of molecules broken at the blocked replication forks.

We report here that DNA double-strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse-field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E. coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild-type E. coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression.

The RepA protein of plasmid pC194 initiates and terminates rolling circle replication. At initiation, it forms a 5'-phosphotyrosyl DNA link, whereas at termination, a glutamate residue directs hydrolytic cleavage of the newly synthesized origin, and the resulting 3'-hydroxyl group undergoes transesterification with the phosphotyrosine link. The protein is thus released from DNA, and the termination is uncoupled from reinitiation of replication. Replacement of the glutamate with tyrosine in RepA altered this mechanism, so that termination occurred by two successive transesterifications and became coupled to reinitiation. This result suggests that various enzymes involved in DNA cleavage and rejoining may have similar mechanistic and evolutionary roots.

We have previously shown that concurrent progression of pBR322 replication and pTac-directed transcription in opposite orientations induces illegitimate recombination events. We tested here the effects of M13 rolling circle replication on the incidence of plasmid deletions. The progression of the M13 replication fork leads to an increase of more than 300-fold in the frequency of transcription-dependent deletion events. pBR322 derivatives carrying the M13 replication origin and a 511 bp transcribed region under the control of the pTac promoter were used. Up to 12% of the plasmid population has sustained deletions within 4 h following the induction of pTac-directed transcription and M13 DNA replication, provided that the two proceed in opposite orientations. We observed that induction of transcription of the whole Escherichia coli lacZ gene (3244 bp) in the direction opposite to M13 replication leads to a fivefold decrease in plasmid copy number within 2 h, which is consistent with the proposal that deletions arise because replication fork progression is impeded. This decrease in parental plasmid copy number leads in turn to an enrichment in deleted plasmid forms. Our data confirm and extend the notion that simultaneous transcription and replication in opposite directions can efficiently promote deletion formation. In addition, this instability may be amplified when the rearranged molecules acquire a replicative advantage.

To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of approximately 4 kb. The replicon was derived from a broad-host-range plasmid, pAM beta 1, which replicates by a unidirectional theta mechanism and its thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and approximately 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.

Linear DNA molecules are subject to degradation by various exonucleases in vivo unless their ends are protected. It has been demonstrated that a specific 8-bp sequence, 5'-GCTGGTGG-3', named Chi, can protect linear double-stranded DNA from the major Escherichia coli exonuclease RecBCD. Chi protects linear replication products of rolling-circle plasmids from RecBCD degradation in vivo, in agreement with observations in vitro. A unique 7-bp sequence, 5'-GCGCGTG-3', is shown to protect similar replication products from degradation in Lactococcus lactis strains but not in more distantly related Gram-positive bacteria. The properties of this sequence in L. lactis correspond to those of a Chi site. Linear plasmid replication products have been detected in numerous prokaryotes, suggesting the widespread existence of short species-specific sequences that preserve linear DNA from extensive degradation by host cell exonucleases.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.

We have analyzed the effect of flanking sequences on Tus-induced replication arrest. pBR322 plasmid derivatives which carry the Escherichia coli replication terminator TerB at different locations were used. Efficiency of the replication arrest was estimated from the plasmid copy number and transformation frequency of tus+ cells. We found that flanking sequences do affect replication arrest efficiency, a weak arrest being correlated with the presence of an AT-rich region which is replicated just before TerB. Some sequences located after the replication terminator can also affect replication termination. We propose that the AT-rich regions might impair binding of the Tus protein to the TerB sequence or facilitate helicase-induced unwinding of DNA and Tus displacement from the TerB site.

Plasmid pAM beta 1 from Enterococcus faecalis uses a unidirectional theta mode of replication. We show here that this replication (i) is dependent on a plasmid-encoded replication protein (Rep) but not on a DNA structure typical for origins of most Rep-dependent plasmids and (ii) is initiated by DNA polymerase I (PolI). pAM beta 1 minimal replicon shares no homology with highly conserved ColE1-type replicons, which use PolI for initiation but do not encode a Rep, or with ColE2 and ColE3 replicons, which require PolI for replication and encode a Rep. We propose that pAM beta 1 and a number of other naturally occurring and closely related plasmids from a distinct plasmid class.

We isolated a mutant of plasmid pUB110 that has the following properties in Bacillus subtilis: (i) it is toxic for recA and add cells, particularly at elevated temperature; (ii) it has a copy number threefold higher than that of the parental plasmid, and the extra copies are present as multimers; and (iii) it can efficiently complement replication of a cmp- satellite plasmid, despite being cmp+. All these properties are due to a single change in the plasmid replication protein, i.e., Gly at position 148 to Glu. These properties of the mutant Rep protein reflect a diminished ability to terminate rolling circle replication. We propose that the Rep protein may have a diminished affinity for the plasmid origin; alternatively, it may be impaired for recognition of the plasmid conformations which distinguish initiation and termination.

We inserted foreign DNA segments into plasmids which replicate by a rolling-circle mechanism in Escherichia coli and observed the appearance of high-molecular-weight plasmid multimers (HMW). This phenomenon, which occurs more frequently with GC-rich segments, depends on the mode of replication of the plasmid and on host homologous recombination functions. We found that (i) HMW are formed upon insertion of a foreign DNA segment into a single-stranded DNA plasmid, whereas the same DNA insert has no such effect on a theta replicon, and (ii) HMW are not present in a recA mutant strain but are found in a lexA (Ind-) mutant. Enzymatic studies allowed us to define the HMW structure as linear double-stranded tandem head-to-tail plasmid repeats. Use of heteroplasmid strains showed that HMW production by one plasmid does not affect another resident plasmid, indicating that no host functions are phenotypically inactivated. This distinguishes our system from the HMW observed with various replicons in the absence of RecBCD enzyme activity. We propose that the role of the foreign insert is to protect the DNA from RecBCD exonuclease attack.

Replication initiated at the bacteriophage M13 origin can be affected by interaction of a properly oriented termination signal terB and the Tus protein. The effect can be alleviated by overproduction of the M13 replication gene protein II.

Hybrids composed of phage M13, plasmid pBR322 and the termination signal of Escherichia coli chromosome replication terB were used to show that arrest of DNA synthesis creates a very efficient deletion hot spot. Up to 80% of deletions occurring in these hybrids had one deletion end-point at terB provided that (i) terB was oriented to arrest M13 and pBR322 leading strand synthesis; and (ii) the host cells contained the Tus protein necessary for arresting DNA synthesis at terB. The position of terB and the flanking sequences had little effect on deletion hot spot activity. About 90% of the deletions at terB ended 5-6 nucleotides in front of the major replication arrest site. We propose two models to account for deletion formation and speculate that many genome rearrangements may be due to the pausing of DNA replication.

Numerous bacterial replicons remain poorly characterized due to difficulties in localization of the replication origin. We have circumvented this problem in the characterization and fine mapping of the origin of plasmid pAM beta 1 by exploiting the Bacillus subtilis termination signal, terC. In terC-containing derivatives, theta-form molecules with two invariant endpoints accumulate. The endpoints, which correspond to plasmid origin and terC, were mapped with single-nucleotide precision. Analysis of the replication intermediates of wild-type molecules by two-dimensional gel electrophoresis confirmed the location of the plasmid origin. Our results demonstrate that pAM beta 1 replication proceeds unidirectionally by a theta mechanism. This work confirms the use of termination signals to localize origins, suggests that termination in B. subtilis occurs by a mechanism similar to that of Escherichia coli and establishes that in addition to rolling circle replicating plasmids, Gram positive bacteria harbour plasmids which replicate by a theta mechanism.

Staphylococcus aureus plasmid pE194 manifests a natural thermosensitivity for replication and can be established in several species, both gram positive and gram negative, thus making it attractive for use as a delivery vector. Like most characterized plasmids of gram-positive bacteria, pE194 generates single-stranded DNA. The direction of pE194 replication is clockwise, as determined by the strandedness of free single-stranded DNA. Significant homology exists between a 50-base-pair sequence in the origin of pE194 and sequences present in plasmids pMV158 (Streptococcus agalactiae), pADB201 (Mycoplasma mycoides), and pSH71 (Lactococcus lactis). We used an initiation-termination reaction, in which pE194 initiates replication at its own origin and is induced to terminate at the related pMV158 sequence, to demonstrate that pE194 replicates by a rolling-circle mechanism; the initiation nick site was localized to an 8-base-pair sequence.

We have inserted a DNA fragment composed of (i) the promoter and the export signal of the Bacillus subtilis levansucrase gene; (ii) the sequence encoding the mature part of the Clostridium thermocellum endoglucanase A gene in a specific site of the B. subtilis chromosome. The insert was flanked by directly repeated pBR322 sequences of 3.9 kb. Plasmid pE194, which has a thermosensitive replication, was integrated adjacent to one of the repeats. When the integrated plasmid was allowed to replicate, the insert and one of the repeats was amplified up to a level of about 250 copies per chromosome. Endoglucanase A was efficiently synthesized in, and secreted from, cells containing the amplified structure, since the heterologous fusion protein was the major extracellular protein in a B. subtilis sacUh strain. The NH2-terminal sequence of the secreted protein revealed three different cleavage sites in the vicinity of the signal peptidase recognition sequence.

Many plasmids from gram-positive bacteria replicate via a single-stranded deoxyribonucleic acid (ssDNA) intermediate, most probably by a rolling-circle mechanism (these plasmids are referred to in this paper as ssDNA plasmids). Their plus and minus origins are physically separated, and replicative initiations are not simultaneous; it is this feature that allows visualization of ssDNA replication intermediates. The insertion of foreign DNA into an ssDNA plasmid may provoke a high frequency of deletions, changes of replicative products to high-molecular-weight forms, segregational loss, and decreased plasmid copy numbers. When an ssDNA plasmid is inserted into the chromosome, both deletions and amplifications may be induced. Both the mode of replication and the copy control mechanism affect the fate of inserted foreign material, usually selecting for its loss. Thus, after having tasted various morsels of DNA, the resulting plasmid stays trim. The features of the ssDNA plasmids seem to be beneficial for their viability and propagation, but not for their use as cloning vectors. However, plasmids replicating via ssDNA intermediates are being exploited to yield insights into the mechanisms of recombination and amplification.

Hybrids composed of phage M13 and plasmid pHV33 were used to study the formation of deletions in Escherichia coli. Eighty to ninety percent of the deletion endpoints were at the position of the nick introduced into the M13 replication origin by the phage gene II protein. This suggests the existence of a novel mechanism of illegitimate recombination.

One S. aureus plasmid coding for tetracycline resistance, pT127, and four plasmids (pC194, pC221, pC223, and pUB112) coding for chloramphenicol resistance have been introduced by transformation into B, subtilis. The plasmids replicate in--and confer antibiotic resistance upon--their new host. These experiments show that the potential for genetic exchange between diverse bacterial species is greater than has been commonly assumed.

Precise excision of Tn10 and related transposons occurs by recombination between directly repeated 9 bp sequences that flank the transposon. The excision, which is a model for a class of illegitimate DNA recombination events, was stimulated 10(6) times by induction of single-stranded DNA synthesis, occurred during conversion of single-stranded DNA to double-stranded form, and entailed no transfer of physical material from parental to progeny molecules. We conclude that it occurred by copy choice DNA recombination and suggest that other illegitimate recombination events may occur by a similar mechanism.

Hybrids between plasmids pC194, pBR322 and the bacteriophage f1 undergo deletions in Escherichia coli. The deletions end most often between nucleotides 1445 and 1446 of pC194. That site probably corresponds to a nick in the replication origin of this plasmid. The localization of the other deletion end appears to be determined by the position of the f1 replication fork. Two models accounting for these data are discussed.

Plasmids pUB110, pC194, pE194, and pT181 are commonly used as cloning vectors in both Bacillus subtilis and Staphylococcus aureus. We report that insertion of foreign DNA into any of these plasmids results in the generation of high-molecular-weight plasmid multimers (HMW) of the recombinant, present as tandem head-to-tail copies. HMW was detected in wild-type B. subtilis and S. aureus strains. The production of HMW depended on the nature of the DNA insertion. Inserts of Escherichia coli DNA, e.g., pBR322 or pUC18, resulted in large amounts of HMW, whereas some inserts of S. aureus DNA of the same size had no effect on plasmid profile. The generation of HMW depended on the mode of plasmid replication; plasmids which replicate via a single-stranded DNA intermediate produced HMW upon foreign DNA insertion, whereas plasmid pAM beta 1, which does not generate single-stranded DNA, did not generate HMW. We propose that HMW is a product of imparied termination of rolling-circle replication and that the impairment is due to the nature of the DNA insertion.

The two replication origins of plasmid pUB110 have been characterized. The site of initiation of DNA replication at the plus origin was mapped to within an 8-base-pair sequence. DNA synthesis initiated at the origin was made to terminate precociously in an inserted sequence of 18 base pairs that is homologous to a sequence in the origin. This suggests that pUB110 replicates as a rolling circle. The minus origin of plasmid pUB110 has been characterized, and the minimal sequence required for function has been determined. As with other minus origins, activity is orientation specific with respect to the direction of replication. Its activity is sensitive to rifampin in vivo, suggesting that RNA polymerase catalyzes single-strand to double-strand conversion. Unlike all other plasmids of gram-positive bacteria thus far described, the pUB110 minus origin is functional in more than one host.

The replication of the single-stranded (ss) DNA plasmid pC194 by the rolling circle mechanism was investigated using chimeric plasmids that possess two pC194 replication origins. One of the origins was intact, whereas the other was either intact or mutated. The origins were activated by inducing synthesis of the pC194 replication protein, under the control of lambda phage pL promoter. Initiation of pC194 replication at one origin and termination at the other generated circular ssDNA molecules smaller than the parental chimeric plasmid. From the nature and the amount of ssDNA circles, the activity of an origin could be assessed. Our results show that (i) the signal for initiation of pC194 replication is more stringent than that for termination; (ii) the sequence and structure of the origin are important for its activity and (iii) successful termination of one replication cycle is not followed by reinitiation of another. This last observation differentiates a ssDNA plasmid (pC194) from a ssDNA phage (phi X174).

A group of small Staphylococcus aureus/Bacillus subtilis plasmids was recently found to replicate via a circular single-stranded DNA intermediate (te Riele et al., 1986a). We show here that a 55 bp region of one such plasmid, pC194, has origin activity when complemented in trans by the plasmid replication protein. This region contains two palindromes, 5 and 14 bp long, and a site nicked by the replication protein. DNA synthesis presumably initiated at the nick in the replication origin can be terminated at an 18 bp sequence homologous to the site of initiation, deriving from another plasmid, pUB110, or synthesized in vitro. This result suggests that, similar to the Escherichia coli single-stranded DNA phages, pC194 replicates as a rolling circle. Interestingly, there is homology between replication origins and replication proteins of pC194 and the phage phi mX174.

Plasmid pC194 exists as circular double-stranded and single-stranded DNA in Bacillus subtilis and Staphylococcus aureus. We report here that the plasmid pHV33, composed of pBR322 and pC194, exists as double- and single-stranded DNA in Escherichia coli, provided that the replication functions of pC194 are intact. Single-stranded pHV33 DNA is converted to double-stranded DNA by complementary strand synthesis probably initiated at rriB, a primosome assembly site present on pBR322. The efficiency of complementary strand synthesis affects the double-stranded copy number, which suggests that single-stranded DNA is a plasmid replication intermediate.

Plasmid pBR322 derives from plasmid ColE1 and does not replicate in Escherichia coli strains lacking DNA polymerase I. Hybrids between pBR322 and a plasmid isolated from Staphylococcus aureus, pC194, replicate in such E. coli strains, provided that the pC194 replication region is intact. Inactivation of the pBR322 replication region does not interfere with the replication of hybrids in E. coli. Hybrids between pBR322 and two other plasmids from S. aureus, pT127 and pUB112, and replicate at the restrictive temperature in E. coli having thermosensitive DNA polymerase I. Similar hybrids involving pC221 and pHV400, plasmids from S. aureus and Bacillus subtilis, respectively, do not replicate under such conditions. These results show that some plasmids from a Gram-positive bacterium, S. aureus, can replicate in a Gram-negative one, E. coli.

The effects of plasmid replication on the frequency of homologous recombination have been investigated. For that purpose Bacillus subtilis strains that carry in their chromosome directly repeated DNA sequences, and an integrated copy of plasmid pE194 either proximal or distal to the repeats, were constructed. The repeat consists either of 3.9 X 10(3) base pBR322 sequences or 2.1 X 10(3) base B. subtilis chromosomal sequences. As plasmid pE194 is naturally thermosensitive for replication, the activity of the replicon could be regulated. Recombination between the repeated sequences was infrequent (about 10(-4) per generation) when the integrated plasmid did not replicate. It was 20 to 450 times higher when the plasmid was allowed to replicate, provided that the repeats were in the proximity of the plasmid. These results show that plasmid replication stimulates DNA recombination.

Replication of plasmid pAMβ1 is initiated by DNA polymerase I (Pol I) and completed by DNA polymerase III holoenzyme contained in the replisome machinery. In this study we report that initiation of DNA replication generates D-loop structures containing the nascent leading strand paired to its template, and that D-loop extension is arrested ≈230bp from the initiation site of DNA synthesis in the presence of the plasmid-encoded resolvase. In vitro and in vivo data suggest that this arrest is caused by a collision between Pol I and the resolvase bound to its target. As the arrested D-loop replication intermediates carry a single-stranded primosome-assembly site, we hypothesize that the biological role of the replication arrest is to limit the region replicated by Pol I and to promote the replacement of Pol I by the replisome in order to initiate concerted synthesis of the leading and lagging strands. [ABSTRACT FROM AUTHOR]