Factors involved in the initiation of phage phi 29 DNA replication in vitro: requirement of the gene 2 product for the formation of the protein p3-dAMP complex.

To study the requirements for the in vitro formation of the protein p3-dAMP complex, the first step in phi29 DNA replication, extracts from B. subtilis infected with phi29 mutants in genes 2, 3, 5, 6 and 17, involved in DNA synthesis, have been used. The formation of the initiation complex is completely dependent on the presence of a functional gene 2 product, in addition to protein p3 and phi29 DNA-protein p3 as template. ATP is also required, although it can be replaced by other nucleotides. The products of genes 5, 6 and 17 do not seem to be needed in the formation of the initiation complex. Inhibitors of the host DNA polymerase III, DNA gyrase or RNA polymerase had no effect on the formation of the protein p3-dAMP complex, suggesting that these proteins are not involved in the initiation of phi29 DNA replication. ddATP or aphidicolin, inhibitors of DNA chain elongation, had also no effect on the formation of the initiation complex.     

Structural and functional analysis of temperature-sensitive mutants of the phage phi 29 DNA polymerase.

The cloning and complete sequencing of gene 2 from four independently isolated temperature-sensitive mutants in the phage phi 29 DNA polymerase (ts2 mutants) is reported. The results obtained indicate that, in vivo, the mutations only affect the initial steps of the replication process. Interestingly, three of these mutations consist in the single amino acid change Ala to Val at position 492 of the protein. The ts2(24) and ts2(98) mutant phi 29 DNA polymerases were expressed, purified and their thermosensitivity was studied at two different steps of DNA replication: 1) protein-primed initiation and 2) elongation of the DNA chain. Whereas the ts2(24) mutation gave rise to a temperature-sensitive phenotype in both reactions, the ts2(98) mutant protein was rather insensitive to the temperature increase. In addition, the ts2(98) mutant protein showed clear differences in the activation by divalent cations. The relationship of these results with structural and functional domains in the phi 29 DNA polymerase are discussed.     

Replication of phage phi 29 DNA in vitro: role of the viral protein p6 in initiation and elongation.

The phi 29 protein p6 stimulates the formation of the protein p3-dAMP initiation complex when added to a minimal system containing the terminal protein p3, the phi 29 DNA polymerase p2 and phi 29 DNA-protein p3 complex, by decreasing about 5 fold the Km value for dATP. In addition, protein p6 stimulates elongation of the p3-dAMP initiation complex. Whereas the effect of protein p6 on initiation is similar with protein p3-containing fragments from the right or left phi 29 DNA ends, the stimulation of elongation is higher with the right than with the left phi 29 DNA terminal fragment, suggesting DNA sequence specificity. The stimulation by protein p6 of the initiation and elongation steps of phi 29 DNA replication does not require the presence of the parental protein p3 at the phi 29 DNA ends. No effect of protein p6 was obtained on the elongation of the template-primer poly(dT)-(dA) 12-18 by the phi 29 DNA polymerase.     

DNA-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 DNA polymerase.

In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5'-dAMP. Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer. The nature of the TP-dAMP linkage was shown to be identical (a O-5'-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication. Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases. However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield. This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site. Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction. According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a "non-DNA-instructed" protein-primed deoxynucleotidylation.     

In vitro initiation of bacteriophage phi 29 and M2 DNA replication: genes required for formation of a complex between the terminal protein and 5'dAMP

Cell-free extracts prepared from phi 29 and M2-infected Bacillus subtilis cells catalyse the formation of complexes between terminal protein and [alpha-32P]-dAMP in the presence of [alpha-32P]-dATP, MgCl2, ATP, and phage DNA with terminal protein covalently linked at both the 5'ends. The complex formation does not take place when proteinase K-treated DNA is added or when uninfected extract is used. The phi 29 complex thus formed is smaller than the M2 complex, primarily due to the different molecular weights of the respective terminal proteins. Extracts prepared from cells infected with suppressor-sensitive mutants of genes 2 or 3 of phi 29 or genes G or E of M2 do not support complex formation. When the pair of extracts of phi 29 or M2-infected cells are mixed, however, formation of the complex takes place as a result of in vitro complementation. These results indicate that the complex formation observed in vitro reflects in vivo initiation of phage DNA replication. The product of gene 2 of phi 29 may be the enzyme that catalyses formation of the complex.     

Primary structure of bacteriophage M2 DNA polymerase: conserved segments within protein-priming DNA polymerases and DNA polymerase I of Escherichia coli

Bacteriophage M2 encodes its own DNA polymerase which catalyses the formation of a primer protein-5'dAMP initiation complex for DNA replication. To understand the relation of structure to function of this 'protein-priming DNA polymerase', we have determined the nucleotide sequence of the M2 DNA polymerase-encoding gene (gene G). The deduced 572-amino acid sequence of M2 DNA polymerase shows 82.3% overall homology to that of phi 29 DNA polymerase. A homology search with the mutation data matrix revealed that six segments (A-F, from the N terminus) of M2 and phi 29 DNA polymerases are homologous with the sequence of Escherichia coli DNA polymerase I (PolI). Segments D and F coincide with the conserved segments of many other DNA polymerases. Therefore, M2 and phi 29 DNA polymerases have structural features, at least in the conserved segments, similar to those of PolI and other DNA polymerases. Based on the homology with PolI and the location of the mutations for aphidicolin resistance and nucleoside analog resistance of M2, phi 29 and herpes simplex virus type-1 DNA polymerases, we propose that segments A-D of the M2 and phi 29 DNA polymerases constitute a structure which forms the cleft for holding template DNA and that segment D is a region for interacting with dNTP.     

Specific recognition of parental terminal protein by DNA polymerase for initiation of protein-primed DNA replication

The linear genome of Bacillus subtilis phage phi29 has a protein covalently linked to the 5' ends, called parental terminal protein (TP), and is replicated using a free TP as primer. The initiation of phage phi29 DNA replication requires the formation of a DNA polymerase/TP complex that recognizes the replication origins located at the genome ends. The DNA polymerase catalyzes the formation of the initiation complex TP-dAMP, and elongation proceeds coupled to strand displacement. The same mechanism is used by the related phage Nf. However, DNA polymerase and TP from phi29 do not initiate the replication of Nf TP-DNA. To address the question of the specificity of origin recognition, we took advantage of the initiation reaction enhancement in the presence of Mn(2+), allowing us to detect initiation activity in heterologous systems in which DNA polymerase, TP, and template TP-DNA are not from the same phage. Initiation was selectively stimulated when DNA polymerase and TP-DNA were from the same phage, strongly suggesting that specific recognition of origins is brought through an interaction between DNA polymerase and parental TP.     

Compartmentalization of phage phi29 DNA replication: interaction between the primer terminal protein and the membrane-associated protein p1

The bacteriophage phi29 replication protein p1 (85 amino acids) is membrane associated in Bacillus subtilis-infected cells. The C-terminal 52 amino acid residues of p1 are sufficient for assembly into protofilament sheet structures. Using chemical cross-linking experiments, we demonstrate here that p1DeltaC43, a C-terminally truncated p1 protein that neither associates with membranes in vivo nor self-interacts in vitro, can interact with the primer terminal protein (TP) in vitro. Like protein p1, plasmid-encoded protein p1DeltaC43 reduces the rate of phi29 DNA replication in vivo in a dosage-dependent manner. We also show that truncated p1 proteins that retain the N-terminal 42 amino acids, when present in excess, interfere with the in vitro formation of the TP.dAMP initiation complex in a reaction that depends on the efficient formation of a primer TP-phi29 DNA polymerase heterodimer. This interference is suppressed by increasing the concentration of either primer TP or phi29 DNA polymerase. We propose a model for initiation of in vivo phi29 DNA replication in which the viral replisome attaches to a membrane-associated p1-based structure.     

Functional domains in the bacteriophage phi 29 terminal protein for interaction with the phi 29 DNA polymerase and with DNA.

Deletion mutants at the amino- and carboxyl-ends of the phi 29 terminal protein, as well as internal deletion and substitution mutants, whose ability to prime the initiation of phi 29 DNA replication was affected to different extent, have been assayed for their capacity to interact with DNA or with the phi 29 DNA polymerase. One DNA binding domain at the amino end of the terminal protein has been mapped. Two regions involved in the binding to the DNA polymerase, an internal region near the amino-terminus and a carboxyl-terminal one, have been also identified. Interaction with both DNA and phi 29 DNA polymerase are required to led to the formation of terminal protein-dAMP initiation complex to start phi 29 DNA replication.     

Characterization of the phage phi 29 protein p5 as a single-stranded DNA binding protein. Function in phi 29 DNA-protein p3 replication.

The phage phi 29 protein p5, required in vivo in the elongation step of phi 29 DNA replication, was highly purified from Escherichia coli cells harbouring a gene 5-containing plasmid and from phi 29-infected Bacillus subtilis. The protein was characterized as the gene 5 product by amino acid analysis and NH2-terminal sequence determination. The purified protein p5 was shown to bind to single-stranded DNA and to protect it against nuclease degradation. No effect of protein p5 was observed either on the formation of the p3-dAMP initiation complex or on the rate of elongation. However, protein p5 greatly stimulated phi 29 DNA-protein p3 replication at incubation times where the replication in the absence of p5 leveled off.     

Effect of NH4+ ions on phi 29 DNA-protein p3 replication: formation of a complex between the terminal protein and the DNA polymerase.

Ammonium ions stimulated the formation of the phi diameter 29 protein p3-dAMP initiation complex by decreasing the Km value for dATP in a purified system containing the viral terminal protein p3, the viral DNA polymerase p2, and the phi 29 DNA-protein p3 complex as a template. In addition, NH4+ ions stimulated the amount of p3-dAMP complex elongation and increased by about twofold the rate of elongation. The stimulatory effect of NH4+ ions on in vitro phi 29 DNA replication is probably related to the formation of a stable complex between the terminal protein and the DNA polymerase, which was detected only in the presence of NH4+ ions.     

Protein-primed DNA replication: a transition between two modes of priming by a unique DNA polymerase.

Phage phi29 from Bacillus subtilis is a paradigm of the protein-primed replication mechanism, in which a single-subunit DNA polymerase is involved in both the specific protein-primed initiation step and normal DNA elongation. To start phi29 DNA replication, the viral DNA polymerase must interact with a free molecule of the viral terminal protein (TP), to prime DNA synthesis once at each phi29 DNA end. The results shown in this paper demonstrate that the DNA polymerase-primer TP heterodimer is not dissociated immediately after initiation. On the contrary, there is a transition stage in which the DNA polymerase synthesizes a five nucleotide-long DNA molecule while complexed with the primer TP, undergoes some structural change during replication of nucleotides 6-9, and finally dissociates from the primer protein when nucleotide 10 is inserted onto the nascent DNA chain. This behaviour probably reflects the polymerase requirement for a DNA primer of a minimum length to efficiently catalyze DNA elongation. The significance of such a limiting transition stage is supported by the finding of abortive replication products consisting of the primer TP linked up to eight nucleotides, detected during in vitro replication of phi29 TP-DNA particularly under conditions that decrease the strand-displacement capacity of phi29 DNA polymerase.     

In vitro synthesis of late bacteriophage phi 29 RNA.

A crude P-100 fraction prepared from Bacillus subtilis 21 min after infection with wild-type phage phi 29 supported the in vitro synthesis of late phi 29 RNA by added RNA polymerase. Synthesis of late RNA was also detected when purified phi 29 DNA was transcribed by RNA polymerase in the presence of an S-150 fraction obtained by lysis of phi 29-infected cells in the presence of 1 M NaCl. Late phi 29 RNA was not synthesized when either the P-100 or the S-150 fraction was prepared from cultures infected with phi 29 having a mutation in gene 4.     

Site-directed mutagenesis in the DNA linking site of bacteriophage phi 29 terminal protein: isolation and characterization of a Ser232----Thr mutant.

By site-directed mutagenesis we have changed the serine residue 232 of the phi 29 terminal protein, involved in the covalent linkage to dAMP for the initiation of replication, into a threonine residue. The mutant terminal protein has been purified to homogeneity and shown to be inactive in the formation of the initiation complex; nevertheless, the mutant protein retains its ability to interact with the phi 29 DNA polymerase and with the DNA. The results obtained indicate a high specificity in the linking site of the terminal protein.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: DNA-gp3 intermediate in in vivo and in vitro assembly

The assembly of phage phi 29 occurs by a single pathway, and DNA-protein (DNA-gp3) has been shown to be an intermediate on the assembly pathway by a highly efficient in vitro complementation. At 30 degrees C, about one-half of the viral DNA synthesized was assembled into mature phage, and the absolute plating efficiency of phi 29 approached unity. DNA packaging at 45 degrees C was comparable to that at 30 degrees C, but the burst size was reduced by one-third. When cells infected with mutant ts3(132) at 30 degrees C to permit DNA synthesis were shifted to 45 degrees C before phage assembly, DNA synthesis ceased and no phage were produced. However, a variable amount of DNA packaging occurred. Superinfection by wild-type phage reinitiated ts3(132) DNA synthesis at 45 degrees C, and if native gp3 was covalently linked to this DNA during superinfection replication, it was effectively packaged and assembled. Treatment of the DNA-gp3 complex with trypsin prevented in vitro maturation of phi 29, although substantial DNA packaging occurred. A functional gp3 linked to the 5' termini of phi 29 DNA is a requirement for effective phage assembly in vivo and in vitro.     

Transition from initiation to elongation in protein-primed phi 29 DNA replication: salt-dependent stimulation by the viral protein p6.

The transition step from the p3-dAMP initiation complex to the first elongated products, p3-(dAMP)2 and p3-(dAMP)3, requires a dATP concentration higher than that needed for the initiation reaction or for the further elongation of the p3-(dAMP)3 complex. The elongation in phi 29 DNA-protein p3 replication in vitro was strongly inhibited by salt. Under inhibitory salt concentration, the viral protein p6 greatly stimulated phi 29 DNA-protein p3 replication. The effect of protein p6 was not on the rate of elongation but on the amount of elongated product, stimulating the transition from initiation to formation of the first elongation products.     

Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication

The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.     

Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex.