Initiation signals for the conversion of single stranded to double stranded DNA forms in the streptococcal plasmid pLS1.

We have characterized a region in the streptococcal plasmid pLS1 located between nucleotides 4103 and 4218 which is a signal involved in the conversion of single stranded intermediates of replication to double stranded plasmid forms. This region has a large axis of dyad symmetry resulting in the formation of a secondary structure as revealed by the location of endonuclease S1-cleavage sites in supercoiled covalently closed circular pLS1 DNA. Deletions affecting this region caused a fivefold reduction in plasmid copy number, plasmid instability and the accumulation of single-stranded DNA intermediates. The conversion signal of pLS1 has homologues in other staphylococcal plasmids, sharing a consensus sequence located in the loop of the signal. Computer assisted analysis showed that the signal detected in pLS1 has a high degree of homology with the complementary strand origin of the Escherichia coli single stranded bacteriophages phi X174 and M13.     

The localization of replication origins on ARS plasmids in S. cerevisiae

Replication intermediates from the yeast 2 microns plasmid and a recombinant plasmid containing the yeast autonomous replication sequence ARS1 have been analyzed by two-dimensional agarose gel electrophoresis. Plasmid replication proceeds through theta-shaped (Cairns) intermediates, terminating in multiply interlocked catenanes that are resolved during S phase to monomer plasmids. Restriction fragments derived from the Cairns forms contain replication forks and bubbles that behave differently from one another when subjected to high voltage and agarose concentrations. The two-dimensional gel patterns observed for different restriction fragments from these two plasmids indicate that in each plasmid there is a single, specific origin of replication that maps, within the limits of our resolution, to the ARS element. Our results strongly support the long-standing assumption that in Saccharomyces cerevisiae an ARS is an origin of replication.     

In vitro recognition of the replication origin of pLS1 and of plasmids of the pLS1 family by the RepB initiator protein.

Rolling-circle replication of plasmid pLS1 is initiated by the plasmid-encoded RepB protein, which has nicking-closing (site-specific DNA strand transferase) enzymatic activity. The leading-strand origin of pLS1 contains two regions, (i) the RepB-binding site, constituted by three directly repeated sequences (iterons or the bind region), and (ii) the sequence where RepB introduces the nick to initiate replication (the nic region). A series of plasmids, belonging to the pLS1 family, show features similar to those of pLS1 and have DNA sequences homologous to the pLS1 nic region. In addition, they all share homologies at the level of their Rep proteins. However, the bind regions of these plasmids are, in general, not conserved. We tested the substrate specificity of purified RepB of pLS1. The RepB protein has a temperature-dependent nicking-closing action on supercoiled pLS1, as well as on recombinant plasmid DNAs harboring the pLS1 nic region. The DNA strand transferase activity of pLS1-encoded RepB was also assayed on two plasmids of the pLS1 family, namely, pE194 and pFX2. DNAs from both plasmids were relaxed by RepB, provided they had a proper degree of supercoiling; i.e., it was necessary to modulate the supercoiling of pE194 DNA to achieve RepB-mediated DNA relaxation. Single-stranded oligonucleotides containing the nic regions of various plasmids belonging to the pLS1 family, including those of pE194 and pFX2, were substrates for RepB. In vitro, the RepB protein does not need to bind to the iterons for its nicking-closing activity.     

Chloroplast DNA replication in vitro: site-specific initiation from preferred templates.

An enzyme system prepared from maize chloroplasts catalyzes the synthesis of DNA from maize chloroplast DNA sequences cloned in bacterial plasmids. Cloned maize chloroplast DNA fragments Bam HI 17' (2470 bp) and Eco RI x (1368 bp) have been shown to be preferred templates for in vitro DNA synthesis catalyzed by pea chloroplast DNA polymerase preparations [Gold et al. (1987) Proc. Natl. Acad. Sci. USA 84, 194-198]. Analysis of replicative intermediates indicates that although the template activity of the recombinant plasmid pZmcBam 17' is substantially greater than that of the pZmcEco x, replication in both cases originates from within a 455 bp region which overlaps the two plasmids. The remaining approximately 1500 basepair portion of maize chloroplast BamHI fragment 17' is not more active because it contains additional origins for replication. The overlapping region shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that contains a replication origin. Replication is shown to proceed bidirectionally within the 455 bp origin region. Recombinant plasmid pZmc 427, which is also active in the in vitro DNA synthesis assay, promoted localized replication initiation within a 1 kbp Bg1II-Eco RI fragment of the chloroplast DNA insert, a region that includes the 3' terminal part of the psbA gene.     

Lagging-Strand Replication from the ssoA Origin of Plasmid pMV158 in Streptococcus pneumoniae: In Vivo and In Vitro Influences of Mutations in Two Conserved ssoA Regions

The streptococcal plasmid pMV158 replicates by the rolling-circle mechanism. One feature of this replication mechanism is the generation of single-stranded DNA intermediates which are converted to double-stranded molecules. Lagging-strand synthesis initiates from the plasmid single-stranded origin, sso. We have used the pMV158-derivative plasmid pLS1 (containing the ssoA type of lagging-strand origin) and a set of pLS1 derivatives with mutations in two conserved regions of the ssoA (the recombination site B [RS_B] and a conserved 6-nucleotide sequence [CS-6]) to identify sequences important for plasmid lagging-strand replication in Streptococcus pneumoniae. Cells containing plasmids with mutations in the RS_B accumulated 30-fold more single-stranded DNA than cells containing plasmids with mutations in the CS-6 sequence. Specificity of lagging-strand synthesis was tested by the development of a new in vitro replication system with pneumococcal cell extracts. Four major initiation sites of lagging-strand DNA synthesis were observed. The specificity of initiation was maintained in plasmids with mutations in the CS-6 region. Mutations in the RS_B region, on the other hand, resulted in the loss of specific initiation of lagging-strand synthesis and also severely reduced the efficiency of replication.     

Replication of the R6K γ origin in vitro: dependence on wt π and hyperactive πS87N protein variant

The π protein of plasmid R6K is involved in control of replication. The aim of this study was to use an in vitro replication system dependent on an R6K-derived γ origin of replication (γori) to compare replication characteristics of wt π and a hyperactive variant of π protein (πS87N; Filutowicz et al., 1994b. Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution. Nucleic Acids Res. 22, 4211–4215). The characteristics of in vitro replication from γori reported in this investigation are as follows: (i) πS87N is considerably more active in comparison to wt π. (ii) Replication proceeds through Cairns-type intermediates and the initiation site and directionality of the fork movement are similar in the presence of both proteins. (iii) Replication forks emanate unidirectionally in the vicinity of the cluster of seven 22-bp direct repeats within γori. (iv) Replication dependent on wt π, but not πS87N, is stimulated up to 1.5-fold by rifampicin.     

Bidirectional replication of the mini-ColE1 plasmid pVH51.

Replicating molecules of the min-ColE1 plasmid pVH51 have been examined by electron microscopy after cleavage with the restriction endonuclease EcoRI. Replication apparently starts at a unique site indistinguishable from the origin of replication used by the parental plasmid ColE1. In contrast to ColE1, the structure of the majority of the replication intermediates was consistent with a bidirectional mode of replication. A minor portion of the molecules appeared to replicate unidirectionally in either direction from the same origin.     

Three regions in the DNA of plasmid pLS1 show sequence-directed static bending.

Three regions showing abnormal electrophoretic mobility, which is an indication of the existence of bends in DNA, have been observed in the DNA of plasmid pLS1. These loci have been characterized by assays designed to detect sequence-directed bending in DNA (temperature-dependence migration and two dimensional electrophoresis). The first region (locus B-1) was located within a fragment that contains a proposed inhibitor countertranscribed RNA (RNAII). The second locus (B-2) contains the plasmid plus origin of replication and the third region (locus B-3) was located in the vicinity of a putative antisense RNA (RNAI) of unknown function. The centres of the first two bent DNA regions were located by circular permutation assays at nucleotides 882 (locus B-1) and 634 (locus B-2). The bend centre of locus B-1 was found to be upstream of the promoter for the putative antisense RNAII. The centre of curvature in locus B-2 was located in the vicinity of the putative promoter of the replication proteins RepA and RepB and of a sequence that has three 11-bp direct repeats. The DNA sequence at this region showed the existence of A.T tracts, with an internal repeat of 10 to 11 base pairs, for five helix turns. A complex curvature in the DNA of pLS1 at locus B-2 that may have a regulatory role in plasmid replication is postulated.     

Replication of the promiscuous plasmid pLSI: a region encompassing the minus origin of replication is associated with stable plasmid inheritance

Deletion of a region of the promiscuous plasmid pLS1 encompassing the initiation signals for the synthesis of the plasmid lagging strand led to plasmid instability in Streptococcus pneumoniae and Bacillus subtilis. This defect could not be alleviated by increasing the number of copies (measured as double-stranded plasmid DNA) to levels similar to those of the wild-type plasmid pLS1. Our results indicate that in the vicinity of, or associated with the single-stranded origin region of pLS1 there is a plasmid component involved in its stable inheritance. Homology was found between the DNA gyrase binding site within the par region of plasmid pSC101 and the pLS1 specific recombination site RS_R.     

Identification of a predominant replication origin in fission yeast.

We have identified five autonomously replicating sequences (ARSs) in a 100 kbp region of the Schizosaccharomyces pombe chromosome II. Analyses of replicative intermediates of the chromosome DNA by neutral/neutral two-dimensional gel electrophoresis demonstrated that at least three of these ARS loci operate as chromosomal replication origins. One of the loci,ori2004, was utilized in almost every cell cycle, while the others were used less frequently. The frequency of initiation from the respective chromosomal replication origin was found to be roughly proportional to the efficiency of autonomous replication of the corresponding ARS plasmid. Replication from ori2004 was initiated within a distinct region almost the same as that for replication of the ARS plasmid. These results showed that the ori2004 region of approximately 3 kbp contains all the cis elements essential for initiation of chromosome replication.     

Trans- and cis-acting elements for the replication of P1 miniplasmids

Replication-deficient mutants of the unit-copy miniplasmid lambda-P1:5R were isolated after hydroxylamine mutagenesis. Complementation tests showed that the majority of these mutants are defective in the production of the repA protein product. Two of these mutants have suppressible nonsense (amber) mutations. The DNA sequence of one of these, repA103, has been determined. The lesion lies within the repA open reading frame, showing that the repA product is essential for plasmid replication. Complementation of deletion mutants of lambda-P1:5R by repA protein showed that the origin of replication lies to the left of repA and that this 300-base-pair origin region is the only portion of the DNA essential for plasmid replication if repA protein is supplied in trans. Six of the 21 hydroxylamine-induced mutants were not complemented by repA. Replication of three of these could be restored by introduction into the plasmid of a wild-type origin region, suggesting that they were origin-defective. The DNA sequence of two mutants was determined. Mutant rep-11 has a 43-base-pair deletion within the incC sequence (incC is a series of five direct repeats of a 19-base-pair sequence known to be involved in the regulation of plasmid replication). The deletion appears to have been generated by homologous recombination between two repeats. Mutant rep-30 has a single base substitution in a region just to the left of incC that destroys one of five G-A-T-C (dam methylation) sites in this region. As lambda-P1:5R is unable to establish itself as a plasmid in a methylase-defective (dam-) strain, it seems probable that methylation of the G-A-T-C sequences is important for origin function. The incC region and the sequences to its left appear to constitute an essential part of the origin of replication.     

Induced bending of plasmid pLS1 DNA by the plasmid-encoded protein RepA

The broad host range streptococcal plasmid pLS1 encodes for a 5.1-kDa repressor protein, RepA. This protein has affinity for DNA (linear or supercoiled) and is translated from the same mRNA as the replication initiator protein RepB. By gel retardation assays, we observed that RepA shows specificity for binding to the plasmid HinfID fragment, which includes the target of the protein. The target of RepA within the plasmid DNA molecule has been located around the plasmid single site ApaLI. This site is included in a region that contains the promoter for the repA and repB genes and is contiguous to the plasmid ori(+). A complex sequence-directed DNA curvature is observed in this region of pLS1. Upon addition of RepA to plasmid linear DNA or to circularly permuted restriction fragments, this intrinsic curvature was greatly enhanced. Thus, a strong RepA-induced bending could be located in the vicinity of the ApaLI site. Visualization of the bent DNA was achieved by electron microscopy of complexes between RepA and plasmid DNA fragments containing the RepA target.     

Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis.

Plasmid rolling circle replication involves generation of single-stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double-stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell-free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I-deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1-ssoA required the entire DNA polymerase I (polymerase and 5'-3' exonuclease activities). ssDNA containing the pLS1-ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase-directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.     

Isolation and characterization of a new plasmid pSpnP1 from a multidrug-resistant clone of Streptococcus pneumoniae

A novel Streptococcus pneumoniae plasmid (pSpnP1; 5413bp) has been isolated from the multidrug-resistant clone Poland(23F)-16, and its complete nucleotide sequence has been determined. Sequence analysis predicted seven co-directional open reading frames and comparative analyses revealed that plasmid pSpnP1 is different to pDP1, the only previously described pneumococcal plasmid, whereas it is highly similar to pSt08, a plasmid from Streptococcus thermophilus. A double-stranded origin for replication similar to the replication origin of the pC194/pUB110 family was located upstream of the putative rep gene (orf2). It also contained a 144-bp region with over 60% identity to the single-stranded origin type A of the Streptococcus agalactiae plasmid pMV158/pLS1. Detection of single-stranded DNA by Southern blot analysis indicated that pSpnP1 replicates via a rolling circle mechanism. Interestingly, the product of orf1 has a putative Zonular occludens toxin conserved domain present in toxigenic strains of Vibrio cholerae. Real-time PCR assays revealed that this ORF was expressed. Hybridization experiments showed that the pSpnP1 replicon was unusual among other examined antibiotic-resistant pneumococcal clones, although the recombinant plasmids based on pSpnP1 were able to replicate in Bacillus subtilis and Lactococcus lactis.