pT181 Replication Research Articles

A Bacillus anthracis -Based In Vitro System Supports Replication of Plasmid pXO2 as Well as Rolling-Circle-Replicating Plasmids

Capsule-encoding virulence plasmid pXO2 of Bacillus anthracis is predicted to replicate by a unidirectional theta-type mechanism. To gain a better understanding of the mechanism of replication of pXO2 and other plasmids in B. anthracis and related organisms, we have developed a cell-free system based on B. anthracis that can faithfully replicate plasmid DNA in vitro. The newly developed system was shown to support the in vitro replication of plasmid pT181, which replicates by the rolling-circle mechanism. We also demonstrate that this system supports the replication of plasmid pXO2 of B. anthracis. Replication of pXO2 required directional transcription through the plasmid origin of replication, and increased transcription through the origin resulted in an increase in plasmid replication.

Genetic and Biochemical Characterization of the Streptococcus pneumoniae PcrA Helicase and Its Role in Plasmid Rolling Circle Replication

PcrA is a chromosomally encoded DNA helicase of gram-positive bacteria involved in replication of rolling circle replicating plasmids. Efficient interaction between PcrA and the plasmid-encoded replication initiator (Rep) protein is considered a requirement for the plasmid to replicate in a given host, and thus, the ability of a Rep protein to interact with heterologous PcrA helicases has been invoked as a determinant of plasmid promiscuity. We characterized transcription of the Streptococcus pneumoniae pcrA gene in its genetic context and studied the biochemical properties of its product, the PcrA(Spn) helicase. Transcription of the pneumococcal pcrA gene was directed by promoter Pa, consisting of an extended -10 box. Promoter Pa also accounted for expression of a second essential gene, radC, which was transcribed with much lower efficiency than pcrA, probably due to the presence of a terminator/attenuator sequence located between the two genes. PcrA(Spn) displayed single-stranded DNA-dependent ATPase activity. PcrA(Spn) showed 5'-->3' and 3'-->5' helicase activities and bound efficiently to partially duplex DNA containing a hairpin structure adjacent to a 6-nucleotide 5' or 3' single-stranded tail and one unpaired (flap) nucleotide in the complementary strand. PcrA(Spn) interacted specifically with RepC, the initiator of staphylococcal plasmid pT181. Although the pneumococcal helicase was able to initiate unwinding of the RepC-nicked pT181 DNA, it was much less processive in this activity than the cognate staphylococcal PcrA protein. Accordingly, PcrA(Spn) was inefficient in in vitro replication of pT181, and perhaps as a consequence, this plasmid could not be established in S. pneumoniae.

Double-stranded origin nicking and replication initiation are coupled in the replication of a rolling circle plasmid, pT181.

The Staphylococcus aureus rolling circle plasmid pT181 initiator RepC is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. RepC/RepC* heterodimer is an inhibitor of replication. However, in order to act effectively, the initiator/inhibitor protein must be stable. We show here that RepC is stable for at least 90 min, which enables it to function effectively as an inhibitor of replication. This finding also allowed us to carry out the two stages in pT181 replication sequentially: first, binding/nicking of the double-strand origin (DSO) by the pT181-encoded RepC, followed by initiation/elongation by the host cell's DNA replication apparatus. The results demonstrate that these two stages in pT181 replication are functionally coupled and that interruptions in this continuous process generate relaxed pT181 DNA that cannot be used as a template for replication.

The PcrA3 mutant binds DNA and interacts with the RepC initiator protein of plasmid pT181 but is defective in its DNA helicase and unwinding activities

Plasmid rolling-circle replication initiates by covalent extension of a nick generated at the plasmid double-strand origin ( dso) by the initiator protein. The RepC initiator protein binds to the plasmid pT181 dso in a sequence-specific manner and recruits the PcrA helicase through a protein–protein interaction. Subsequently, PcrA unwinds DNA at the nick site followed by replication by DNA polymerase III. The pcrA3 mutant of Staphylococcus aureus has previously been shown to be defective in plasmid pT181 replication. Suppressor mutations in the repC initiator gene have been isolated that allow pT181 replication in the pcrA3 mutant. One such suppressor mutant contains a D57Y change in the RepC protein. To identify the nature of the defect in PcrA3, we have purified this mutant protein and studied its biochemical activities. Our results show that while PcrA3 retains its DNA binding activity, it is defective in its helicase and RepC-dependent pT181 DNA unwinding activities. We have also purified the RepC D57Y mutant and shown that it is similar in its biochemical activities to wild-type RepC. RepC D57Y supported plasmid pT181 replication in cell-free extracts made from wild-type S. aureus but not from the pcrA3 mutant. We also demonstrate that both wild-type RepC and its D57Y mutant are capable of a direct physical interaction with both wild-type PcrA and the PcrA3 mutant. Our results suggest that the inability of PcrA3 to support pT181 replication is unlikely to be due to its inability to interact with RepC. Rather, it is likely that a defect in its helicase activity is responsible for its inability to replicate the pT181 plasmid.

Biochemical Characterization of the Staphylococcus aureus PcrA Helicase and Its Role in Plasmid Rolling Circle Replication

Previous genetic studies have suggested that a putative chromosome-encoded helicase, PcrA, is required for the rolling circle replication of plasmid pT181 in Staphylococcus aureus. We have overexpressed and purified the staphylococcal PcrA protein and studied its biochemical properties in vitro. Purified PcrA helicase supported the in vitro replication of plasmid pT181. It had ATPase activity that was stimulated in the presence of single-stranded DNA. Unlike many replicative helicases, PcrA was highly active as a 5' --> 3' helicase and had a weaker 3' --> 5' helicase activity. The RepC initiator protein encoded by pT181 nicks at the origin of replication and becomes covalently attached to the 5' end of the DNA. The 3' OH end at the nick then serves as a primer for displacement synthesis. PcrA helicase showed an origin-specific unwinding activity with supercoiled plasmid pT181 DNA that had been nicked at the origin by RepC. We also provide direct evidence for a protein-protein interaction between PcrA and RepC proteins. Our results are consistent with a model in which the PcrA helicase is targeted to the pT181 origin through a protein-protein interaction with RepC and facilitates the movement of the replisome by initiating unwinding from the RepC-generated nick.

Role of the Double-Strand Origin Cruciform in pT181 Replication

pT181 is a small rolling-circle plasmid from Staphylococcus aureus whose initiator protein, RepC, melts the plasmid's double-strand origin (DSO) and extrudes a cruciform involving IR II, a palindrome flanking the initiation nick site. We have hypothesized that the cruciform is required for initiation, providing a single-stranded region for the assembly of the replisome (R. Jin et al., 1997, EMBOJ. 16, 4456–4566). In this study, we have tested the requirement for cruciform extrusion by disrupting the symmetry of the IR II palindrome or by increasing its length. The modified DSOs were tested for replication with RepC in trans. Rather surprisingly, disruption of the IR II symmetry had no detectable effect on replication or on competitivity of the modified DSO, though plasmids with IR II disrupted were less efficiently relaxed than the wild type by RepC. However, in conjunction with IR II disruption, modification of the tight RepC binding site IR III blocked replication. These results define two key elements of the pT181 initiation mechanism—the IR II conformation and the RepC binding site (IR III)—and they indicate that pT181 replication initiation is sufficiently robust to be able to compensate for significant modifications in the configuration of the DSO.

PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling-circle replication.

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.

Double-stranded origin nicking and replication initiation are coupled in the replication of a rolling circle plasmid, pT181

The Staphylococcus aureus rolling circle plasmid pT181 initiator RepC is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC *. RepC/RepC * heterodimer is an inhibitor of replication. However, in order to act effectively, the initiator/inhibitor protein must be stable. We show here that RepC is stable for at least 90 min, which enables it to function effectively as an inhibitor of replication. This finding also allowed us to carry out the two stages in pT181 replication sequentially: first, binding/nicking of the double-strand origin (DSO) by the pT181-encoded RepC, followed by initiation/elongation by the host cell's DNA replication apparatus. The results demonstrate that these two stages in pT181 replication are functionally coupled and that interruptions in this continuous process generate relaxed pT181 DNA that cannot be used as a template for replication.

Sequence requirements for the termination of rolling-circle replication of plasmid pT181.

Most small multicopy plasmids of Gram-positive bacteria and many in Gram-negative bacteria replicate by a rolling-circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single-stranded bacteriophages of Escherichia coli have origin-specific nicking-closing activities that are required for the initiation and termination of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nick site is located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.

The inactivated plasmid inititator protein RepC/RepC* may have a regulatory role.

During replication of the plasmid pT181, the initiator protein RepC is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. Here we show that during in vitro replication, RepC* is radioactively labeled, suggesting that the source of the RepC* oligodeoxynucleotide is the newly synthesized pT181 DNA. The RepC/RepC* heterodimer retains its ability to bind the pT181 double-strand origin and, therefore, it may act as a competitive inhibitor of the RepC homodimer during replication.

Modification of the plasmid initiator protein RepC active site during replication.

pT181 is a Staphylococcus aureus rolling circle replicating plasmid whose copy number is controlled by regulating the synthesis and activity of the initiator protein, RepC*. The RepC* dimer is modified during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC. To purify RepC, RepC was expressed in S. aureus as a fusion protein with a polyhistidine tail. The histidine-tagged RepC retains its initiation and topoisomerase activities in vitro. His-tagged RepC/RepC and RepC/RepC* were purified in a two-step procedure. Peptide mapping, mass spectrometric analysis and protein sequencing of purified RepC and RepC* were carried out, and both proteins appeared identical, except that the peptide containing the RepC active site tyrosine used in nicking activity was absent when the purified RepC* sample was analyzed. The absence of the active site in RepC* suggests that this site was modified during replication. The results provide the first direct biochemical evidence that RepC* is a modified form of RepC, and support a model in which RepC replication of pT181 leaves RepC with an oligonucleotide blocking the active site of one of its subunits.

Modification of the plasmid initiator protein RepC active site during replication

pT181 is a Staphylococcus aureus rolling circle replicating plasmid whose copy number is controlled by regulating the synthesis and activity of the initiator protein, RepC. The RepC dimer is modified during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC ∗. To purify RepC ∗, RepC was expressed in S. aureus as a fusion protein with a polyhistidine tail. The histidine-tagged RepC retains its initiation and topoisomerase activities in vitro. Histagged RepC/RepC and RepC/RepC ∗ were purified in a two-step procedure. Peptide mapping, mass spectrometric analysis and protein sequencing of purified RepC and RepC ∗ were carried out, and both proteins appeared identical, except that the peptide containing the RepC active site tyrosine used in nicking activity was absent when the purified RepC ∗ sample was analyzed. The absence of the active site in RepC ∗ suggests that this site was modified during replication. The results provide the first direct biochemical evidence that RepC ∗ is a modified form of RepC, and support a model in which RepC replication of pT181 leaves RepC with an oligonucleotide blocking the active site of one of its subunits.

An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181.

pT181 and related plasmids of gram-positive bacteria replicate by a rolling-circle mechanism. The replication initiator protein of pT181, RepC, has origin-specific nicking-closing activities. Replication of the plasmid pT181 leading strand initiates by covalent extension of the RepC-generated nick, and the origin of replication contains signals for both initiation and termination of DNA replication. We have investigated the sequence requirements for the initiation and termination steps by using plasmids containing two pT181 origins. In vitro replication experiments showed that 18- and 24-bp synthetic oligonucleotides containing the RepC nick site were active in the termination of replication. However, initiation of replication required a larger region which also includes the RepC binding site. Plasmids containing the 18- and 24-bp region were also found to be nicked by the RepC protein. Our results demonstrate that sequence requirements for initiation and termination of pT181 replication overlap, but while the RepC binding site is required for initiation, it is dispensable for termination.

Plasmid pT181 replication is decreased at high levels of RepC per plasmid copy

The replication of staphylococcal plasmid pT181 is indirectly controlled at the level of the synthesis of its replication initiator, RepC. As a result, high levels of RepC synthesis per plasmid copy were expected to lead to autocatalytic plasmid replication, which secondarily would affect host physiology. Surprisingly, RepC overexpression was found to lead to a rapid decrease in pT181 copy number and replication rate. These effects depended on the ratio of RepC to the pT181 replication origin rather than on the absolute amount of RepC in the cell. In a wild-type host, the increase in RepC/plasmid copy also inhibited chromosome replication and cell division. The changes in host physiology did not play any role in the decrease in pT181 replication caused by RepC overexpression since pT181 replication responded in the same way in a host mutant insensitive to the effects of RepC induction. These results suggest that pT181, the prototype of an entire class of plasmids from Gram-positive bacteria, responds to overexpression of its replication initiator by a decrease in plasmid replication.

Replication Enhancer Requirement for Recognition of Heterologous Replication Origin by an Initiator Protein

In the pT181 plasmid family, the replication initiation protein (Rep) encoded by each plasmid recognizes only its cognate origin, unless the Rep protein is expressed at abnormally high levels. Heterologous recognition of the origin of the pC221 plasmid by the RepC protein of the pT181 plasmid requires that cmp, the pT181 replication enhancer, be present on the same plasmid as the origin of replication. These findings indicate that cmp has a role in the specificity of Rep- ori recognition and support the model that cmp facilitates the formation/stabilization of the RepC-origin complex.

Localization of the start sites of lagging‐strand replication of rolling‐circle plasmids from Gram‐positive bacteria

A number of small, multicopy plasmids from Gram-positive bacteria replicate by an asymmetric rolling-circle mechanism. Previous studies with several of these plasmids have identified a palindromic sequence, SSOA, that acts as the single-strand origin (SSO) for the replication of the lagging-strand DNA. Although not all the SSOA sequences share DNA sequence homology, they are structurally very similar. We have used an in vitro system to study the lagging-strand replication of several plasmids from Gram-positive bacteria using the SSOA sequences of pT181, pE194 and pSN2 as representative of three different groups of Staphylococcus aureus plasmids. In addition, we have investigated the lagging-strand replication of the pUB110 plasmid that contains an alternative single-strand origin, SSOU. Our results confirm that RNA polymerase is involved in lagging-strand synthesis from both SSOA and SSOU-type lagging-strand origins. Interestingly, while initiation of lagging-strand DNA synthesis of pUB110 occurred predominantly at a single position within SSOU, replication of pT181, pSN2 and pE194 plasmids initiated at multiple positions from SSOA.

Replication-specific conversion of the Staphylococcus aureus pT181 initiator protein from an active homodimer to an inactive heterodimer.

The Staphylococcus aureus rolling circle plasmid pT181 regulates its replication by controlling the synthesis of its initiator protein RepC. RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. We analyzed RepC and RepC* in four classes of mutants: plasmid copy number mutants, two classes of RepC mutants affecting different portions of the protein and oriC (origin) mutants. We have found that in the cell with wild-type RepC there are similar relative amounts of RepC and RepC*, regardless of copy number, and that the conversion of RepC to RepC* is replication dependent. Genetic and biochemical evidence is presented that RepC functions as a dimer and that during replication the RepC homodimer is converted to the RepC/RepC* heterodimer.

Plasmids of the pT181 family show replication-specific initiator protein modification.

The rolling circle plasmids of Staphylococcus aureus regulate their replication by controlling initiator (Rep) protein synthesis. It was demonstrated recently that the pT181 initiator protein RepC is inactivated during pT181 replication by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC* (A. Rasooly and R. P. Novick, Science, 262:1048-1050). We establish here that this initiator modification occurs with four other members of the pT181 family and that it occurs in Bacillus subtilis as well as S. aureus. These results suggest that Rep conversion to Rep* is probably universal among plasmids of the pT181 family and is not host dependent.

Characterization of the Staphylococcus aureus chromosomal gene pcrA, identified by mutations affecting plasmid pT181 replication.

The Staphylococcus aureus chromosomal gene pcrA, identified by mutations, such as pcrA3, that affect plasmid pT181 replication, has been cloned and sequenced. The pcrA gene encodes a protein with significant similarity (40% identity) to two Escherichia coli helicases: the helicase II encoded by the uvrD gene and the Rep helicase. The pcrA3 mutation was found to be a C to T transition leading to a threonine to isoleucine substitution at amino acid residue 61 of the protein. The pcrA gene seems to belong to an operon containing at least one other gene, tentatively named pcrB, upstream from pcrA. The PcrA protein was shown to be essential for cell viability and overproduction has deleterious effects on the host and plasmid replication.

Plasmid pT181-linked suppressors of the Staphylococcus aureus pcrA3 chromosomal mutation.

Plasmid pT181 replication is affected in hosts carrying the chromosomal pcrA3 mutation, resulting in significantly lower plasmid copy numbers. Mutations suppressing this effect have been isolated and characterized. The suppressor mutations were found to map in the plasmid repC gene and manifested pcrA allele specificity, suggesting the existence of a direct RepC-PcrA interaction.