The Escherichia coli primosome can translocate actively in either direction along a DNA strand

The primosome is a mobile multiprotein DNA replication-priming apparatus that requires seven Escherichia coli proteins (replication factor Y (protein n'), proteins n and n", and the products of the dnaB, dnaC, dnaT, and dnaG genes) for assembly at a specific site (termed a primosome assembly site) on single-stranded DNA binding protein-coated single-stranded DNA. Two of the protein components of the primosome have intrinsic DNA helicase activity. The DNA B protein acts in the 5'----3' direction, whereas factor Y acts in the 3'----5' direction. The primosome complex has DNA helicase activity when present at a replication fork in conjunction with the DNA polymerase III holoenzyme. In this report, evidence is presented that the multiprotein primosome per se can act as a DNA helicase in the absence of the DNA polymerase III holoenzyme. The primosome DNA helicase activity can be manifested in either direction along the DNA strand. The directionality of the primosome DNA helicase activity is modulated by the concentration and type of nucleoside triphosphate present in the reaction mixture. This DNA helicase activity requires all the preprimosomal proteins (the primosomal proteins minus the dnaG-encoded primase). Preprimosome complexes must assemble at a primosome assembly site in order to be loaded onto the single-stranded DNA and act subsequently as a DNA helicase. The 5'----3' primosome DNA helicase activity requires a 3' single-stranded tail on the fragment to be displaced, while the 3'----5' activity does not require a 5' single-stranded tail on the fragment to be displaced. Multienzyme preprimosomes moving in either direction are capable of associating with the primase to form complete primosomes that can synthesize RNA primers.     

Early steps of Bacillus subtilis primosome assembly

Primosomes are nucleoprotein assemblies designed for the activation of DNA replication forks. Their primary role is to recruit the replicative helicase onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. PriA is conserved in bacteria, but its primosomal partners are not. In Bacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB and DnaD proteins unravels their role in primosome assembly. They are both multimeric and bind individually to DNA. Furthermore, DnaD stimulates DnaB binding activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork.     

Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate.

PriA and other primosome assembly proteins of Escherichia coli recruit the major replicative helicase DnaB for replisome assembly during bacteriophage Mu transposition and replication. MuA transposase catalyzes the transfer of Mu ends to target DNA, forming a potential replication fork that provides the assembly site for the replisome. However, this fork lacks the single-stranded DNA needed to load DnaB. Although no pre-existing primosome assembly sites that bind PriA were found within the Mu end sequences, PriA was able to bind to the forked DNA structure created by MuA. The helicase activity of PriA could then open the duplex to create the DnaB binding site. In a tightly coupled reaction on synthetic forked substrates, PriA promoted both the unwinding of the lagging strand arm and preprimosome assembly to load DnaB onto the lagging strand template. PriA apparently translocated 3' to 5' along the lagging strand template until sufficient single-stranded DNA was exposed for binding of DnaB, which then translocated 5' to 3' in the opposite direction. Mutant PriA lacking helicase activity was unable to promote this process, and loss of PriA helicase impaired Mu DNA replication in vivo and in vitro. This suggests that the opening of the duplex by PriA helicase is a critical step in the initiation of Mu DNA replication. Concerted helicase and primosome assembly functions would allow PriA to act as initiator on recombination intermediates and stalled replication forks. As part of the replisome, PriA may act as a mobile initiator that minimizes interruptions in chromosomal replication.     

Dissecting the functional role of PriA protein-catalysed primosome assembly in Escherichia coli DNA replication

The multi-functional PriA protein of Escherichia coli (formerly replication factor Y or protein n') serves to guide the ordered assembly of the primosome, a mobile multiprotein replication priming/helicase complex. Primosome assembly is essential for bacteriophage OX174 complementary DNA strand synthesis and ColE1-type plasmid replication reconstituted in vitro with purified proteins. The biochemical activities of the primosome suggest that it can fulfill the primase/helicase requirement on the lagging-strand DNA template during cellular DNA replication. However, reconstruction in vitro of DNA replication of small plasmids containing the E. coli origin of DNA replication (oriC) does not require the complete complement of primosomal proteins. Thus, the extent to which PriA-catalysed primosome assembly participates in chromosomal replication has remained unclear. The recent isolation of the genes encoding PriA, PriB (protein n), PriC (protein n"), and DnaT (protein i) has provided the necessary tools for addressing this issue. The phenotype of mutations in these genes, and other results described in this review, suggest that assembly of the primosome catalysed by PriA does in fact contribute at some stage to normal cellular DNA replication. A model for primososme-catalysed reactivation of a dysfunctional replication fork is discussed.     

Prokaryotic DNA replication mechanisms

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.     

Effects of the bacteriophage T4 gene 41 and gene 32 proteins on RNA primer synthesis: coupling of leading- and lagging-strand DNA synthesis at a replication fork

We have demonstrated previously that the template sequences 5'-GTT-3' and 5'-GCT-3' serve as necessary and sufficient signals for the initiation of new DNA chains that start with pentaribonucleotide primers of sequence pppApCpNpNpN or pppGpCpNpNpN, respectively. Normally, the complete T4 primosome, consisting of the T4 gene 41 (DNA helicase) and gene 61 (primase) proteins, is required to produce RNA primers. However, a high concentration of the 61 protein alone can prime DNA chain starts from the GCT sites [Cha, T.-A., & Alberts, B. M. (1986) J. Biol. Chem. 261, 7001-7010]. We show here that the 61 protein can catalyze a single-stranded DNA template-dependent reaction in which the dimers pppApC and pppGpC are the major products and much longer oligomers of various lengths are minor ones. Further addition of the 41 protein is needed to form a primosome that catalyzes efficient synthesis of the physiologically relevant pentaribonucleotides that are responsible for the de novo DNA chain starts on the lagging strand of a replication fork. The helicase activity of the 41 protein is necessary and sufficient to ensure a high rate and processivity of DNA synthesis on the leading strand [Cha, T.-A., & Alberts, B. M. (1989) J. Biol. Chem. 264, 12220-12225]. Coupling an RNA primase to this helicase in the primosome therefore coordinates the leading- and lagging-strand DNA syntheses at a DNA replication fork. Our experiments reveal that the addition of the T4 helix-destabilizing protein (the gene 32 protein) is required to confine the synthesis of RNA primers to those sites where they are used to start an Okazaki fragment, causing many potential priming sites to be passed by the primosome without triggering primer synthesis.     

Leading and lagging strand synthesis at the replication fork of bacteriophage T7. Distinct properties of T7 gene 4 protein as a helicase and primase

Reactions at the replication fork of bacteriophage T7 have been reconstituted in vitro on a preformed replication fork. A minimum of three proteins is required to catalyze leading and lagging strand synthesis. The T7 gene 4 protein, which exists in two forms of molecular weight 56,000 and 63,000, provides helicase and primase activities. A tight complex of the T7 gene 5 protein and Escherichia coli thioredoxin provides DNA polymerase activity. Gene 4 protein and DNA polymerase catalyze processive leading strand synthesis. Gene 4 protein molecules serving as helicase remain bound to the template as leading strand synthesis proceeds greater than 40 kilobases. Primer synthesis for lagging strand synthesis is catalyzed by additional gene 4 protein molecules that undergo multiple association/dissociation steps to catalyze multiple rounds of primer synthesis. The smaller molecular weight form of gene 4 protein has been purified from an equimolar mixture of both forms. Removal of the large form results in the loss of primase activity but not of helicase activity. Submolar amounts of the large form present in a mixture of both forms are sufficient to restore high specific activity of primase characteristic of an equimolar mixture of both forms. These results suggest that the gene 4 primase is an oligomer which is composed of both molecular weight forms. The large form may be the distributive component of the primase which dissociates from the template after each round of primer synthesis.     

The effect of the T7 and Escherichia coli DNA-binding proteins at the replication fork of bacteriophage T7

In this paper we compare the effect of single-stranded DNA-binding proteins of bacteriophage T7 (gene 2.5 protein) and of Escherichia coli (SSB) at the T7 replication fork. The T7 gene 4 protein acts processively as helicase to promote leading strand synthesis and distributively as primase to initiate lagging strand synthesis by T7 DNA polymerase. On a nicked double-stranded template, the formation of a replication fork requires partial strand displacement so that gene 4 protein may bind to the displaced strand and unwind the helix catalytically. Both the T7 gene 2.5 protein and E. coli SSB act stoichiometrically to promote this initial strand displacement step. Once initiated, processive leading strand synthesis is not greatly stimulated by the single-stranded DNA-binding proteins. However, the T7 gene 2.5 protein, but not E. coli SSB, increases the frequency of initiation of lagging strand synthesis by greater than 10-fold. The results suggest a specific interaction of the T7 gene 2.5 protein with the T7 replication apparatus.     

DnaB, DnaD and DnaI proteins are components of the Bacillus subtilis replication restart primosome.

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.     

Conserved residues of the C-terminal p16 domain of primase are involved in modulating the activity of the bacterial primosome

The bacterial primosome comprises the replicative homo-hexameric ring helicase DnaB and the primase DnaG. It is an integral component of the replisome as it unwinds the parental DNA duplex to allow progression of the replication fork, synthesizes the initiation primers at the replication origin, oriC , and the primers required for Okazaki fragment synthesis during lagging strand replication. The interaction between the two component proteins is mediated by a distinct C-terminal domain (p16) of the primase. Both proteins mutually regulate each other's activities and a putative network of conserved residues has been proposed to mediate these effects. We have targeted 10 residues from this network. To investigate the functional contributions of these residues to the primase, ATPase and helicase activities of the primosome, we have used site-directed mutagenesis and in vitro functional assays. Five of these residues (E464, H494, R495, Y548 and R555) exhibited some functional significance while the remaining five (E483, R484, E506, D512 and E530) exhibited no effects. E464 participates in functional modulation of the primase activity, whereas H494, R495 and R555 participate in allosteric functional modulation of the ATPase and/or helicase activities. Y548 contributes directly to the structural interaction with DnaB.     

The crystal structure of the bifunctional primase-helicase of bacteriophage T7

Within minutes after infecting Escherichia coli, bacteriophage T7 synthesizes many copies of its genomic DNA. The lynchpin of the T7 replication system is a bifunctional primase-helicase that unwinds duplex DNA at the replication fork while initiating the synthesis of Okazaki fragments on the lagging strand. We have determined a 3.45 A crystal structure of the T7 primase-helicase that shows an articulated arrangement of the primase and helicase sites. The crystallized primase-helicase is a heptamer with a crown-like shape, reflecting an intimate packing of helicase domains into a ring that is topped with loosely arrayed primase domains. This heptameric isoform can accommodate double-stranded DNA in its central channel, which nicely explains its recently described DNA remodeling activity. The double-jointed structure of the primase-helicase permits a free range of motion for the primase and helicase domains that suggests how the continuous unwinding of DNA at the replication fork can be periodically coupled to Okazaki fragment synthesis.     

Escherichia coli and Bacillus subtilis PriA proteins essential for recombination-dependent DNA replication: involvement of ATPase/helicase activity of PriA for inducible stable DNA replication

The E. coli PriA protein, a DEXH-type DNA helicase with unique zinc finger-like motifs interrupting the helicase domains, is an essential component of the phiX174-type primosome and plays critical roles in RecA-dependent inducible and constitutive stable DNA replication (iSDR and cSDR, respectively) as well as in recombination-dependent repair of double-stranded DNA breaks. B. subtilis PriA (BsPriA) protein contains the conserved helicase domains as well as zinc finger-like motifs with 34% overall identity with the E. coli counterpart. We overexpressed and purified BsPriA and examined its biochemical properties. BsPriA binds specifically to both n'-pas (primosome assembly site) and D-loop and hydrolyzes ATP in the presence of n'-pas albeit with a specific activity about 30% of that of E. coli PriA. However, it is not capable of supporting n'-pas-dependent replication in vitro, nor is it able to support ColE1-type plasmid replication in vivo which requires the function of the phiX174-type primosome. We also show that a zinc finger mutant is not able to support recombination-dependent DNA replication, as measured by the level of iSDR after a period of thymine starvation, nor wild-type level of growth, cell morphology and UV resistance. Unexpectedly, we discovered that an ATPase-deficient mutant (K230D) is not able to support iSDR to a full extent, although it can restore normal growth rate and UV resistance as well as non-filamentous morphology in priA1::kan mutant. K230D was previously reported to be fully functional in assembly of the phiX174-type primosome at a single-stranded n'-pas. Our results indicate that ATP hydrolysis/ helicase activity of PriA may be specifically required for DNA replication from recombination intermediates in vivo.     

Functional difference between the two oppositely oriented priming signals essential for the initiation of the broad host-range plasmid RSF1010 DNA replication.

The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the origin of vegetative DNA replication (oriV). If either ssiA or ssiB was deleted or inverted, the RSF1010 miniplasmids containing engineered oriVs were maintained at low copy numbers, replicated abnormally as dimers, and accumulated specific single strands in the Escherichia coli strain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins. Interestingly, an additional intracellular supply of the Sog primase (the sog gene product of plasmid CoIIb-P9) reversed the replication deficiency of these miniplasmids with respect to all three aspects described above. These were also true for the RSF1010 miniplasmids in which either ssiA or ssiB was replaced by the primosome assembly site (PAS) or by the G4-type ssi signal (G site). Furthermore, comparative analysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespective of their types, ssi signals conducting the initiation of DNA chain elongation away from the iterons were functionally more important than ones in the inverted orientation. We consider that this functional difference reflects the inherent properties of the initiation mechanism of RSF1010 DNA replication.     

Identification of Subunit Binding Positions on a Model Fork and Displacements That Occur during Sequential Assembly of the Escherichia coli Primosome

When replication stalls and forks disassemble, the restart primosome is required to reload the replicative helicase so that chromosomal replication can be reinitiated. We have taken a photo-cross-linking approach, using model replication forks containing a phenyl diazirine placed at single locations, to determine the positions of primosomal protein binding and changes in interactions that occur during the assembly reaction. This approach revealed a novel mode for single-stranded DNA-binding protein (SSB)-DNA binding, in which SSB interacts with both the leading and lagging single-strand segments and the parental duplex of the fork. Cross-linking to a novel region within SSB is observed only when it is bound to forked structures. This binding mode is also followed by PriB. PriA binds to the fork, excluding SSB and PriB, interacting with the primer terminus, single-stranded leading and lagging strands and duplex in immediate proximity of the fork. SSB binds to flanking single-stranded segments distal to the fork in the presence of PriA. The addition of PriB or DnaT to a PriA-SSB-fork complex does not lead to cross-linking or displacement, suggesting that their association is through protein-protein interactions at early stages of the reaction. Upon addition of DnaC and the DnaB helicase in the presence of ATPγS, helicase is assembled, leading to contacts within the duplex region on the tracking (lagging) strand and strong contacts with the displaced leading single strand near the fork. PriA is displaced from DNA upon helicase assembly.     

The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate

A DNA replication system was developed that could generate rolling-circle DNA molecules in vitro in amounts that permitted kinetic analyses of the movement of the replication forks. Two artificial primer-template DNA substrates were used to study DNA synthesis catalyzed by the DNA polymerase III holoenzyme in the presence of either the preprimosomal proteins (the primosomal proteins minus the DNA G primase) and the Escherichia coli single-stranded DNA binding protein or the DNA B helicase alone. Helicase activities have recently been demonstrated to be associated with the primosome, a mobile multiprotein priming apparatus that requires seven E. coli proteins (replication factor Y (protein n'), proteins n and n', and the products of the dnaB, dnaC, dnaG, and dnaT genes) for assembly, and with the DNA B protein. Consistent with a rolling-circle mechanism in which a helicase activity permitted extensive (-) strand DNA synthesis on a (+) single-stranded, circular DNA template, the major DNA products formed were multigenome-length, single-stranded, linear molecules. The replication forks assembled with either the preprimosome or the DNA B helicase moved at the same rate (approximately 730 nucleotides/s) at 30 degrees C and possessed apparent processivities in the range of 50,000-150,000 nucleotides. The single-stranded DNA binding protein was not required to maintain this high rate of movement in the case of leading strand DNA synthesis catalyzed by the DNA polymerase III holoenzyme and the DNA B helicase.     

Mechanisms of primer RNA synthesis and D-loop/R-loop-dependent DNA replication in Escherichia coli

In DNA replication, DNA chains are generally initiated from small pieces of ribonucleotides attached to DNA templates. These ‘primers’ are synthesized by various enzymatic mechanisms in Escherichia coli. Studies on primer RNA synthesis on single-stranded DNA templates containing specific ‘priming signals’ revealed the presence of two distinct modes, ie immobile and mobile priming. The former includes primer RNA synthesis by primase encoded by dnaG and by RNA polymerase containing a σ⁷⁰ subunit. Priming is initiated at a specific site in immobile priming. Novel immobile priming signals were identified from various plasmid replicaons, some of which function in initiation of the leading strand synthesis. The latter, on the other hand, involves a protein complex, primosome, which contains DnaB, the replicative helicase for E coli chromosomal replication. Utilizing the energy fueled by ATP hydrolysis of DnaB protein, primosomes are able to translocate on a template DNA and primase synthesizes primer RNAs at multiple sites. Two distinct primosomes. DnaA-dependent primosome supports normal chromosomal identified, which are differentially utilized for E coli chromosomal replication. Whereas DnaA-dependent primosome supports normal chromosomal replication from oriC, the PriA-dependent primosome functions in oriC-independent chromosomal replication observed in DNA-damaged cells or cells lacking RNaseH activity. In oriC-independent replication, PriA protein may recognize the D- or R-loop structure, respectively, to initiate assembly of a primosome which mediates primer RNA synthesis and replication fork progression.     

Initiation of lagging-strand synthesis for pBR322 plasmid DNA replication in vitro is dependent on primosomal protein i encoded by dnaT

The role of the primosome assembly and protein n' recognition site in replication of pBR322 plasmid was examined. The following evidence indicates that the primosome is involved in lagging-strand synthesis of pBR322 plasmid replication in vitro. Early replicative intermediates with newly synthesized leading strand, approximately 1 kilobase pair long, immediately downstream of the replication origin accumulate in products synthesized in extracts from a dnaT strain that lacks primosomal protein i or in wild-type extracts supplemented with anti-protein i antibody. These intermediates are converted efficiently into full-length DNA by addition of purified protein i. Consistent with the previously proposed role of the primosome (Arai, K. and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 69-73), an n' site on the lagging strand, but not on the leading strand, is required for efficient replication of the plasmid in vitro. Plasmids lacking an n' site on the lagging strand replicate only to a limited extent in vitro and early replicative intermediates carrying nascent leading strands are accumulated, although a portion of the intermediates complete replication to yield full-length DNA. The latter reaction is completely inhibited by addition of anti-protein i antibody. Insertion of the n' site of phage phi X174 into pBR322 plasmids lacking lagging-strand n' sites restores the replicative ability of the mutant plasmid comparable to that of the wild-type plasmid. These results indicate that protein i is essential for lagging-strand synthesis of pBR322 plasmid in vitro and that it may play an important role in the priming events as a part of either an n' site-dependent primosome or an n' site-independent, as yet unidentified, priming complex.     

DnaG-dependent priming signals can substitute for the two essential DNA initiation signals in oriV of the broad host-range plasmid RSF1010.

Broad host-range plasmid RSF1010 contains in the oriV region two DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010), which are essential for plasmid replication. Each of ssiA and ssiB could be substituted functionally by either of the two G4-type (DnaG-dependent) priming signals, the oric of bacteriophage G4 and an ssi signal from plasmid pSY343 (an R1 plasmid derivative). Functions of the chimeric oriVs of RSF1010 thus constructed were dependent on the RSF1010-specific replication proteins, RepA, RepB' and RepC. When both of ssiA and ssiB were replaced by the G4-type ssi signals, functions of the chimeric oriVs were no longer dependent on RepB' (RSF1010-specific DNA primase). The replication activities of the chimeric oriVs of RSF1010 were not influenced markedly by the type of heterologous priming signals they contained. It is conceivable that DNA replication of RSF1010 does not need the priming mechanism for lagging strand synthesis and proceeds by the strand displacement mechanism.     

Escherichia coli PriA helicase: fork binding orients the helicase to unwind the lagging strand side of arrested replication forks

Escherichia coli PriA is a primosome assembly protein with 3' to 5' helicase activity whose apparent function is to promote resumption of DNA synthesis following replication-fork arrest. Here, we describe how initiation of helicase activity on DNA forks is influenced by both fork structure and by single-strand DNA-binding protein. PriA could recognize and unwind forked substrates where one or both arms were primarily duplex, and PriA required a small (two bases or larger) single-stranded gap at the fork in order to initiate unwinding. The helicase was most active on substrates with a duplex lagging-strand arm and a single-stranded leading-strand arm. On this substrate, PriA was capable of translocating on either the leading or lagging strands to unwind the duplex ahead of the fork or the lagging-strand duplex, respectively. Fork-specific binding apparently orients the helicase domain to unwind the lagging-strand duplex. Binding of single-strand-binding protein to forked templates could inhibit unwinding of the duplex ahead of the fork but not unwinding of the lagging-strand duplex or translocation on the lagging-strand template. While single-strand-binding protein could inhibit binding of PriA to the minimal, unforked DNA substrates, it could not inhibit PriA binding to forked substrates. In the cell, single-strand-binding protein and fork structure may direct PriA helicase to translocate along the lagging-strand template of forked structures such that the primosome is specifically assembled on that DNA strand.     

Are single-stranded circles intermediates in plasmid DNA replication?

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.