Interference between M13 and oriM13 plasmids is mediated by a replication enhancer sequence near the viral strand origin

The origin of replication for the viral strand of bacteriophage M13 DNA is contained within a 507 base-pair intergenic region of the phage chromosome. The viral strand origin is defined as the specific site at which the M13 gene II protein nicks the duplex replicative form of M13 DNA to initiate rolling-circle synthesis of progeny viral DNA. Using in vitro techniques we have constructed deletion mutations in M13 DNA at the unique AvaI site which is located 45 nucleotides away on the 3' side of the gene II protein nicking site. This deletion analysis has identified a sequence near the viral strand origin that is required for efficient replication of the M13 genome. We refer to this part of the intergenic region as a "replication enhancer" sequence. We have also studied the function of this sequence in chimeric pBR322-M13 plasmids and found that plasmids carrying both the viral strand origin and the replication enhancer sequence interfere with M13 phage replication. Based upon these findings we propose a model for the mechanism of action of the replication enhancer sequence involving binding of the M13 gene II protein.     

Deletion analysis of the cloned replication origin region from bacteriophage M13.

A cloned 270-nucleotide fragment from the origin region of the M13 duplex replicative form DNA confers an M13-dependent replication mechanism upon the plasmid vector pBR322. This M13 insert permits M13 helper-dependent replication of the hybrid plasmid in polA cells which are unable to replicate the pBR322 replicon alone. Using in vitro techniques, we have constructed several plasmids containing deletions in the M13 DNa insert. The endpoints of these deletions have been determined by DNA sequence analysis and correlated with the transformation and replication properties of each plasmid. Characterization of these deletion plasmids allows the following conclusions. (i) The initiation site for M13 viral strand replication is required for helper-dependent propagation of the chimeric plasmid. (ii) A DNA sequence in the M13 insert, localized between 89 and 129 nucleotides from the viral strand initiation site, is necessary for efficient transformation of polA cells. A chimeric plasmid containing the viral strand initiation site, but lacking this additional 40 nucleotide M13 sequence, transforms helper-infected cells at a frequency approximately 10(4)-fold less than that of plasmids containing this additional DNA segment. (iii) The entire M13 complementary strand origin can be deleted without affecting M13-dependent transformation by the hybrid plasmids. We propose a model in which replication of one strand of duplex chimera initiates by nicking at the gene II protein nicking site in the viral strand of the M13 insert, followed by asymmetric single-strand synthesis. Initiation of the complementary strand possibly occurs within plasmid sequences.     

DNA sequences which support activities of the bacteriophage phi X174 gene A protein

The DNA sequence of 30 nucleotides which surrounds the origin of viral strand DNA replication is highly conserved amongst the icosahedral single-stranded DNA bacteriophages. The A gene of these phages encodes a protein which is required for initiation and termination of viral strand DNA synthesis and acts as a nicking-closing activity specifically within this 30-nucleotide sequence. A system of purified Escherichia coli host proteins and phi X174 gene A protein has been developed which specifically replicates in vitro the viral strand of phi X174 from RF (replicative form) I template DNA and yields single-stranded circular DNA products (RF leads to SS(c) DNA replication system). Recombinant plasmids carrying inserts derived from phage phi X174 or G4 DNA which range in length from 49 to 1175 base pairs and contain the 30-nucleotide conserved sequence have been shown to support phi X A protein-dependent DNA synthesis in vitro in this replication system. We report here that insertion of the 30-nucleotide sequence alone into pBR322 allows the resulting recombinant plasmids to support phi X A protein-dependent in vitro DNA synthesis as efficiently as phi X174 template DNA in the RF leads to SS(c) replication system. The 30-nucleotide sequence functions as a fully wild type DNA replication origin as determined by the rate of DNA synthesis and the structure of resulting DNA products. Furthermore, the DNA sequence requirements for nicking of RF I DNA by the phi X A protein and for supporting replication origin function have been partially separated. Homology to positions 1, 29, and 30 of the 30-nucleotide conserved sequence are not required for cleavage of RF I DNA by the A protein; homology to position 1 but not 29 or 30 is required for efficient DNA replication.     

Specificity of RepC protein in plasmid pT181 DNA replication

The plasmid pT181 of Staphylococcus aureus consists of 4437 base pairs and encodes resistance to tetracycline. Initiation of pT181 DNA replication specifically requires the plasmid-encoded initiator protein, RepC. The initiator protein binds specifically to a 32-base pair sequence within the pT181 origin of replication. RepC protein also has a nicking-closing activity that is specific for the pT181 origin. Replication of pT181 initiates by covalent extension of the nick and proceeds by a rolling circle mechanism. Two other small, multicopy plasmids pC221 and pS194 belong to the pT181 family and have common structural organization and replication properties. The replication proteins and replication origins of these plasmids have extensive sequence homologies, although they belong to different incompatibility groups. In spite of this homology, the replication proteins and replication origins of these three plasmids do not show any cross-reactivity in vivo. We have carried out a series of in vitro experiments to determine the specificity of pT181-encoded initiator protein, RepC. DNA binding experiments showed that although the binding of RepC to the pT181 origin was very efficient, little or no binding was seen with pC221 and pS194 origins. The nicking-closing activity of RepC was found to be equally efficient with the pC221 and pS194 plasmids. The plasmids pC221 and pS194 replicated efficiently in a RepC-dependent in vitro system. However, replication of these plasmids was greatly reduced in the presence of a competing pT181 origin. The results presented here suggest that nicking-closing by RepC at the origin is not sufficient for maximal replication and that tight binding of RepC to the origin plays an important role in the initiation of DNA replication.     

Plasmid rolling-circle replication: highlights of two decades of research

This review provides a historical perspective of the major findings that contributed to our current understanding of plasmid rolling-circle (RC) replication. Rolling-circle-replicating (RCR) plasmids were discovered approximately 20 years ago. The first of the RCR plasmids to be identified were native to Gram-positive bacteria, but later such plasmids were also identified in Gram-negative bacteria and in archaea. Further studies revealed mechanistic similarities in the replication of RCR plasmids and the single-stranded DNA bacteriophages of Escherichia coli, although there were important differences as well. Three important elements, a gene encoding the initiator protein, the double strand origin, and the single strand origin, are contained in all RCR plasmids. The initiator proteins typically contain a domain involved in their sequence-specific binding to the double strand origin and a domain that nicks within the double strand origin and generates the primer for DNA replication. The double strand origins include the start-site of leading strand synthesis and contain sequences that are bound and nicked by the initiator proteins. The single strand origins are required for synthesis of the lagging strand of RCR plasmids. The single strand origins are non-coding regions that are strand-specific, and contain extensive secondary structures. This minireview will highlight the major findings in the study of plasmid RC replication over the past twenty years. Regulation of replication of RCR plasmids will not be included since it is the subject of another review.     

A comparison of the origin of replication of pSa with R6K

Summary The plasmid pOri3 is a derivative of the origin of replication of pSa. Replication is defective as a result of a truncated repA gene, the product of which is required for plasmid replication. The defective replication is complemented by the presence of the intact repA gene of pSa, or by the presence of the plasmid R6K. The basis of this complementation has been examined by comparing the nucleotide sequence of the origin of pSa with that of R6K. A 13 base pair sequence present twice in the origin of pSa has homology with a 13 base pair sequence that is present fourteen times in the origin of R6K. These sequences may be the binding sites for the initiator proteins of these two plasmids. The location of these binding sites relative to the genes for the initiator proteins suggests that an autoregulatory control mechanism for the synthesis of the initiator proteins may also play a role in the control of plasmid copy number.     

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of ColE plasmid DNA.

The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and phi X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles phi X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of phi X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.     

Studies on the role of the phi X174 gene A protein in phi X174 viral strand synthesis. III. Replication of DNA containing two viral replication origins

Supercoiled plasmid bearing two wild-type phi X origin sequences on the same strand supported the phi X A protein-dependent in vitro formation of two smaller single-stranded circles, the lengths of which were equivalent to the distance between the two origins. Additional double origin plasmids were utilized to determine whether origins defective in the initial nicking event (initiation) could support circularization (termination). In all cases tested, the presence of a mutant origin on the same strand with a wild-type origin affected the level of replication in a manner consistent with the previously determined activity of the mutant origin. When a functional mutant origin was present on the same strand as a wild-type origin, the efficiency of replication and the DNA products formed were almost identical to those of the plasmid containing two wild-type origins. Plasmid DNA bearing both a wild-type origin and a mutant origin that did not support phi X A protein binding or nicking activity, on the other hand, supported efficient DNA synthesis of only full-length circular products, indicating that the origin defective for initiation was incapable of supporting termination. In contrast, the presence of a wild-type origin and an origin that did bind the phi X A protein but was not cleaved resulted in a marked decrease in DNA synthesis along with the production of only full-length products. This suggests that the phi X A protein stalls when it encounters a sequence to which it can bind but cannot cleave. Replication of double origin plasmids containing one functional phi X origin on each strand of the supercoiled DNA was also examined. With such templates, synthesis from the wild-type origin predominated, indicating preferential cleavage of the intact origin sequence. Replication of such substrates also produced a number of aberrant structures, the properties of which suggested that interstrand exchange of the phi X A protein had occurred.     

Characterization of the herpes simplex virus origin binding protein interaction with OriS.

The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717). Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS. Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site. This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM. Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less. OBP binds with high affinity to duplex DNA containing mismatched base pairs. This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands. For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair. For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove. In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.     

Initiation signals for complementary strand DNA synthesis on single-stranded plasmid DNA.

The bacteriophage 0X174 origin for (+) strand DNA synthesis, when inserted in a plasmid, is in vivo a substrate for the initiator A protein, that is produced by infecting phages. The result of this interaction is the packaging of single-stranded plasmid DNA into preformed phage coats. These plasmid particles can transduce 0X-sensitive cells; however, the transduction efficiency depends strongly on the presence in the packaged DNA strand of an initiation signal for complementary strand DNA synthesis. A plasmid with the complementary (-) strand origin of 0X inserted in the same strand as the viral (+) origin transduces 50-100 times more efficient than the same plasmid without the (-) origin of 0X. The transduction efficiency of such a particle is comparable to the infection efficiency of the phage particle. It is shown that in this system the 0X (-) origin can be replaced by the complementary strand origins of the bacteriophages G4 and M13. We have used this system to isolate sequences, from E. coli plasmids (pACYC177, CloDF13, miniF and OriC) and from the E. coli chromosome that can function as initiation signals for the conversion of single-stranded plasmid DNA to double-stranded DNA. All isolated origins were found to be dependent for their activity on the dnaB, dnaC and dnaG proteins. We conclude that these signals were all primosome-dependent origins and that primosome priming is the major mechanism for initiation of the lagging strand DNA synthesis in E. coli. The assembly of the primosome depends on the sequence-specific interaction of the n' protein with single-stranded DNA. We have used the isolated sequences to deduce a consensus recognition sequence for the n' protein. The role of a possible secondary structure in this sequence is discussed.     

The functional origin of bacteriophage f1 DNA replication. Its signals and domains

The origin of DNA replication of bacteriophage f1 functions as a signal, not only for initiation of viral strand synthesis, but also for its termination. Viral (plus) strand synthesis initiates and terminates at a specific site (plus origin) that is recognized and nicked by the viral gene II protein. Mutational analysis of the 5' side (upstream) of the origin of plus strand replication of phage f1 led us to postulate the existence of a set of overlapping functional domains. These included ones for strand nicking, and initiation and termination of DNA synthesis. Mutational analysis of the 3' side (downstream) of the origin has verified the existence of these domains and determined their extent. The results indicate that the f1 "functional origin" can be divided into two domains: (1) a "core region", about 40 nucleotides long, that is absolutely required for plus strand synthesis and contains three distinct but partially overlapping signals, (a) the gene II protein recognition sequence, which is necessary both for plus strand initiation and termination, (b) the termination signal, which extends for eight more nucleotides on the 5' side of the gene II protein recognition sequence, (c) the initiation signal that extends for about ten more nucleotides on the 3' side of the gene II protein recognition sequence; (2) a "secondary region", 100 nucleotides long, required exclusively for plus strand initiation. Disruption of the secondary region does not completely abolish the functionality of the f1 origin but does drastically reduce it (1% residual biological activity). We discuss a possible explanation of the fact that this region can be interrupted (e.g. f1, M13 cloning vectors) by large insertions of foreign DNA without significantly affecting replication.     

Six amino acids determine the sequence-specific DNA binding and replication specificity of the initiator proteins of the pT181 family.

The replication of pT181 and related plasmids of Staphylococcus aureus proceeds by a rolling circle mechanisms. The initiator proteins encoded by the plasmids of the pT181 family have sequence-specific DNA binding and topoisomerase activities. These proteins nick one strand of the DNA at the origin of replication. The free 3'-hydroxyl end at the nick is then used as a primer for the replication of the leading strand of the DNA. Although these initiator proteins are highly homologous, they show specificity in DNA binding and replication for their cognate DNAs. In this study, we have generated hybrid initiator proteins and studied their various biochemical activities in vitro. Our results show that 6 amino acids are sufficient to determine the DNA binding and replication specificities of such initiator proteins.     

The gene II proteins of the filamentous phages IKe and Ff (M13, fd and f1) are not functionally interchangeable during viral strand replication.

Gene II protein is the only phage-encoded protein required for the double-stranded DNA replication of the distantly related filamentous phages IKe and Ff (M13, fd and f1). Complementation studies have demonstrated that, despite a significant degree of homology between the nucleotide sequences of the gene II's of IKe and Ff and the core's (domains A) of their viral strand replication origins, the biological functions of the gene II proteins are not interchangeable. The specificity of these proteins is not determined by the nucleotide sequence (domain B) which is required for efficient initiation of viral strand replication of Ff. In fact, our data indicate that a sequence with a similar function as domain B in Ff does not form part of the viral strand replication origin of IKe.     

Structure and function of the adenovirus origin of replication

Efficient initiation of adenovirus DNA replication requires the presence of specific terminal nucleotide sequences that collectively constitute the viral origin of replication. Using plasmids with deletions or base substitutions in a cloned segment of DNA derived from the terminus of the adenovirus 2 genome, we have demonstrated that the origin contains two functionally distinct regions. The first 18 bp of the viral genome are sufficient to support a limited degree of initiation. However, the presence of a sequence in the region between nucleotides 19 and 67 greatly enhances the efficiency of the initiation reaction. This region contains a specific binding site for a protein present in uninfected cells (KD = 2 X 10(-11) M). The bound protein protects the DNA segment between base pairs 19 and 43 from attack by DNAase I. Studies with deletion mutants indicate that binding of the cellular protein is responsible for the enhancement of initiation.     

Specificity of origin recognition by replication initiator protein in plasmids of the pT181 family is determined by a six amino acid residue element.

We have investigated the specificity of replication origin recognition by the initiator proteins of a set of six closely related Staphylococcus aureus plasmids, the pT181 family. These plasmids replicate by an asymmetric rolling-circle mechanism using plasmid-coded initiators that nick the replication origins and form a phosphotyrosine bond at the 5' nick terminus. Five of the plasmids are in different incompatibility groups and their initiator proteins do not cross-complement the cloned origins of any but their own plasmid. One pair is weakly incompatible and their initiator proteins and origins do cross-complement for replication in vivo. This pattern of cross-reactivity led to the prediction that the determinant of specificity would correspond to a homologously positioned set of six residues in the C-terminal domain of the protein, some 80 residues away from the active site tyrosine, that are divergent for all of the compatible plasmids and identical for the incompatible pair. Site-directed mutagenesis was used to exchange these six residues among three pairs of plasmids and these exchanges brought about the predicted switching of origin recognition specificity. Single substitution within this six residue set reduced or eliminated the activity of the protein but did not alter the origin recognition specificity. These six and flanking residues cannot form an amphipathic alpha-helix nor do they conform to the classical helix-turn-helix or other known DNA binding motifs. A novel type of interaction is suggested in which the protein binds to its recognition site, bends and melts the DNA, and causes or enhances the extrusion of an adjacent cruciform containing the nick site. This configuration would juxtapose the nicking target and the active site tyrosine residue and would unwind the highly G + C-rich replication origin.     

Genes and regulatory sequences of bacteriophage phi X174

Fragments of the DNA of bacteriophage phi X174 were inserted in the plasmids pACYC177 and pBR322, in order to test the in vivo effects of separate phage genes and regulatory sequences. The phi X174 inserts were identified by recombination and complementation with phage mutants, followed by restriction enzyme analysis. The genes B, C, F and G can be maintained stably in the cell even when there is efficient expression of these viral genes. Recombinant plasmids with the complete genes D and E can only be maintained when the expression of these genes is completely blocked. Expression of complete H and J genes could not yet be demonstrated. The intact gene A was apparently lethal for the host cell, as it was never found in the recombinants. The genes F and G are expressed, even when they are not preceded by one of the well characterized viral or plasmid promoter sequences. Screening of the nucleotide sequence of phi X174 gives two promoter-like sequences just in front of the two genes. Viral sequences with replication signals (the phi X174 (+) origin of replication, the initiation site for complementary strand synthesis and the incompatibility sequence) appeared to be functional also when inserted in recombinant plasmids. A plasmid with the phi X (+) origin can be forced to a rolling circle mode of replication. The A protein produced by infecting phages works in trans on the cloned viral origin. The (-) origin can function as initiation signal for complementary strand synthesis during transduction of single-stranded plasmid DNA. The intracellular presence of the incompatibility sequence on a plasmid prevents propagation of infecting phages.     

Effect of phi X C protein on leading strand DNA synthesis in the phi X174 replication pathway

The influence of the bacteriophage phi X174 (phi X) C protein on the replication of bacteriophage phi X174 DNA has been examined. This small viral protein, which is required for the packaging of phi X DNA into proheads, inhibits leading strand DNA synthesis. The inhibitory effect of the phi X C protein requires a DNA template bearing an intact 30-base pair (bp) phi X origin of DNA replication that is the target site recognized by the phi X A protein. Removal of nucleotides from the 3' end of this 30-bp conserved origin sequence prevents the inhibitory effects of the phi X C protein. Leading strand replication of supercoiled DNA substrates containing the wild-type phi X replication origin results in the production of single-stranded circular DNA as well as the formation of small amounts of multimeric and sigma structures. These aberrant products are formed when the termination and reinitiation steps of the replication pathway reactions are skipped as the replication fork moves through the origin sequence. Replication carried out in the presence of the phi X C protein leads to a marked decrease in these aberrant structures. While the exact mechanism of action of the phi X C protein is not clear, the results presented here suggest that the phi X C protein slows the movement of the replication fork through the 30-bp origin sequence, thereby increasing the fidelity of the termination and reinitiation reactions. In keeping with the requirement for the phi X C protein for efficient packaging of progeny phi X DNA into proheads, the phi X C protein-mediated inhibition of leading strand synthesis is reversed by the addition of proteins essential for phi X bacteriophage formation. Incubation of plasmid DNA substrates bearing mutant 30 base pair phi X origin sequences in the complete packaging system results in the in vitro packaging and production of infectious particles in a manner consistent with the replication activity of the origin under study.     

Molecular analysis and characterization of a broad-host-range plasmid, pEP2.

Plasmid pEP2 was found to encode a protein, RepA, which is essential and rate limiting for its replication in Escherichia coli and Corynebacterium pseudotuberculosis. Mutations which altered the rate of synthesis of this protein in E. coli affected the copy number and segregational stability of pEP2 in the two hosts. RepA contains 483 amino acid residues and has the calculated molecular weight of 53,925. It shows 45% amino acid residue identity with open reading frame ORF2 of pSR1, a plasmid isolated from Corynebacterium glutamicum (J. A. C. Archer and A. J. Sinskey, J. Gen. Microbiol. 139:1753-1759, 1993). Plasmid pEP2 was shown to accumulate single-stranded DNA corresponding to the RepA coding strand during its replication in E. coli and C. pseudotuberculosis, suggesting that it may replicate by a rolling circle mechanism. However, RepA has no significant sequence homology with the replication initiator proteins of plasmids known to use this mode of replication.     

A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication.

We recently reported that human immunodeficiency virus type 1 (HIV-1) unintegrated linear DNA displays a discontinuity in its plus strand, precisely defined by a second copy of the polypurine tract (PPT) located near the middle of the genome (P. Charneau and F. Clavel, J. Virol. 65:2415-2421, 1991). This central PPT appears to determine a second initiation site for retrovirus DNA plus-strand synthesis. We show here that mutations replacing purines by pyrimidines in the HIV-1 central PPT, which do not modify the overlapping amino acid sequence, are able to significantly slow down viral growth as they reduce plus-strand origin at the center of the genome. One of these mutations, introducing four pyrimidines, results in a 2-week delay in viral growth in CEM cells and abolishes plus-strand origin at the central PPT. The introduction in this mutant of a wild-type copy of the PPT at a different site creates a new plus-strand origin at that site. This new origin also determines the end of the upstream plus-strand segment, probably as a consequence of limited strand displacement-synthesis. Our findings further demonstrate the role of PPTs as initiation sites for the synthesis of the retroviral DNA plus strand and demonstrate the importance of a second such origin for efficient HIV replication in vitro.