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.     

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.     

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.     

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.     

Function of the C-terminus of phi29 DNA polymerase in DNA and terminal protein binding

The thumb subdomain, located in various family B DNA polymerases in the C-terminal region, has been shown in their crystal structures to move upon binding of DNA, changing its conformation to nearly completely wrap around the DNA. It has therefore been involved in DNA binding. In agreement with this, partial proteolysis studies of 29 DNA polymerase have shown that the accessibility of the cleavage sites located in their C-terminal region is reduced in the presence of DNA or terminal protein (TP), indicating that a conformational change occurs in this region upon substrate binding and suggesting that this region might be involved in DNA and TP binding. Therefore, we have studied the role of the C-terminus of 29 DNA polymerase by deletion of the last 13 residues of this enzyme. This fragment includes a previously defined region conserved in family B DNA polymerases. The resulting DNA polymerase Δ13 was strongly affected in DNA binding, resulting in a distributive replication activity. Additionally, the capacity of the truncated polymerase to interact with TP was strongly reduced and its initiation activity was very low. On the other hand, its nucleotide binding affinity and its fidelity were not affected. We propose that the C-terminal 13 amino acids of 29 DNA polymerase are involved in DNA binding and in a stable interaction with the initiator protein TP, playing an important role in the intrinsic processivity of this enzyme during polymerization.     

Deletions at the N terminus of bacteriophage phi 29 protein p6: DNA binding and activity in phi 29 DNA replication

Deletions corresponding to the first 5 or 13 amino acids (aa), not counting the initial Met, have been introduced into the N terminus of the phage phi 29 protein p6. The activity of such proteins in the in vitro phi 29 DNA replication system, their capacity to interact with the phi 29 DNA ends, and their interference with the wild type (wt) protein p6 activity have been studied. The initiation activity of protein p6 decreased considerably when 5 as were deleted and was undetectable when 13 aa were removed. The mutant proteins were unable to specifically interact with the phi 29 DNA ends. These results indicate the need of an intact N terminus for the activity of protein p6. However, such N-truncated proteins inhibited both the specific binding of the wt protein p6 to the phi 29 DNA ends and its activity in phi 29 DNA replication.     

Involvement of phage phi29 DNA polymerase and terminal protein subdomains in conferring specificity during initiation of protein-primed DNA replication

To initiate ϕ29 DNA replication, the DNA polymerase has to form a complex with the homologous primer terminal protein (TP) that further recognizes the replication origins of the homologous TP-DNA placed at both ends of the linear genome. By means of chimerical proteins, constructed by swapping the priming domain of the related ϕ29 and GA-1 TPs, we show that DNA polymerase can form catalytically active heterodimers exclusively with that chimerical TP containing the N-terminal part of the homologous TP, suggesting that the interaction between the polymerase TPR-1 subdomain and the TP N-terminal part is the one mainly responsible for the specificity between both proteins. We also show that the TP N-terminal part assists the proper binding of the priming domain at the polymerase active site. Additionally, a chimerical ϕ29 DNA polymerase containing the GA-1 TPR-1 subdomain could use GA-1 TP, but only in the presence of ϕ29 TP-DNA as template, indicating that parental TP recognition is mainly accomplished by the DNA polymerase. The sequential events occurring during initiation of bacteriophage protein-primed DNA replication are proposed.     

Regions at the carboxyl end of bacteriophage phi 29 protein p6 required for DNA binding and activity in phi 29 DNA replication.

Series of deletions corresponding to the carboxyl end of the phage phi 29 protein p6 have been constructed and their activity in the initiation of phi 29 DNA replication and their capacity to interact with the phi 29 DNA ends have been studied. Determination of the activity of the deletion mutants in phi 29 DNA replication indicated the dispensability of the 14 carboxy-terminal amino acids of the protein. The activity of protein p6 decreased with deletions from 23 to 39 amino acids and was undetectable when 44 amino acids were removed. A similar behaviour was obtained when the interaction of the mutant proteins with the phi 29 DNA ends was analyzed. These results indicate that the stimulation of phi 29 DNA replication by protein p6 requires a specific binding to the phi 29 DNA ends.     

The Bacillus subtilis phage phi 29 protein p16.7, involved in phi 29 DNA replication, is a membrane-localized single-stranded DNA-binding protein.

The functional role of the phi 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage phi 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA). Protein p16.7A was found to be an ssDNA-binding protein. In addition, by direct and functional analysis we show that protein p16.7A binds to the stretches of ssDNA of the phi 29 DNA replication intermediates. Properties of protein p16.7A were compared with those of the phi 29-encoded single-stranded DNA-binding protein p5. The results obtained show that both proteins have different, non-overlapping functions. The likely role of p16.7 in attaching phi 29 DNA replication intermediates to the membrane of the infected cell is discussed. Homologues of gene 16.7 are present in phi 29-related phages, suggesting that the proposed role of p16.7 is conserved in this family of phages.     

Characterization of the bacteriophage phi29-encoded protein p16.7: a membrane protein involved in phage DNA replication

An early expressed operon, located at the right end of the linear bacteriophage phi29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in phi29-related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N-terminally located transmembrane-spanning domain is required for its membrane localization. The variant p16.7A, in which the N-terminal membrane anchor was replaced by a histidine-tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a phi29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivo phi29 DNA replication is discussed.     

The integral membrane protein p16.7 organizes in vivo phi29 DNA replication through interaction with both the terminal protein and ssDNA

Remarkably little is known about the in vivo organization of membrane-associated prokaryotic DNA replication or the proteins involved. We have studied this fundamental process using the Bacillus subtilis phage phi29 as a model system. Previously, we demonstrated that the phi29-encoded dimeric integral membrane protein p16.7 binds to ssDNA and is involved in the organization of membrane-associated phi29 DNA replication. Here we demonstrate that p16.7 forms multimers, both in vitro and in vivo, and interacts with the phi29 terminal protein. In addition, we show that in vitro multimerization is enhanced in the presence of ssDNA and that the C-terminal region of p16.7 is required for multimerization but not for ssDNA binding or interaction with the terminal protein. Moreover, we provide evidence that the ability of p16.7 to form multimers is crucial for its ssDNA-binding mode. These and previous results indicate that p16.7 encompasses four distinct modules. An integrated model of the structural and functional domains of p16.7 in relation to the organization of in vivo phi29 DNA replication is presented.     

Initiation of phage phi 29 DNA replication by the terminal protein modified at the carboxyl end

A mutant at the carboxyl end of the terminal protein, p3, of phage phi 29 DNA has been constructed by inserting an containing the stop translation codon TGA in the three possible reading frames, immediately downstream of a phage phi 29 DNA fragment coding for all but the last five amino acids of protein p3. The activity in the formation of the p3-dAMP initiation complex in vitro of this mutant as well as another one previously isolated, also mutated at the carboxyl end, have been tested. The results obtained suggest that an intact carboxyl end in the phage phi 29 terminal protein is essential for its normal primer function in DNA replication.     

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.     

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.     

Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication.     

Mechanism of stimulation of DNA replication by bacteriophage phi 29 single-stranded DNA-binding protein p5

Protein p5 is a Bacillus subtilis phage phi 29-encoded protein required for phi 29 DNA replication in vivo. Protein p5 has single-stranded DNA binding (SSB) capacity and stimulates in vitro DNA replication severalfold when phi 29 DNA polymerase is used to replicate either the natural phi 29 DNA template or primed M13 single-stranded DNA (ssDNA). Furthermore, other SSB proteins, including Escherichia coli SSB, T4 gp32, adenovirus DNA-binding protein, and human replication factor A, can functionally substitute for protein p5. The stimulatory effect of phi 29 protein p5 is not due to an increase of the DNA replication rate. When both phi 29 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, phi 29 DNA replication is strongly inhibited. This inhibition is fully overcome by adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably phi 29 DNA polymerase, which has a strong affinity for ssDNA. Electron microscopy demonstrates that protein p5 binds to M13 ssDNA forming saturated complexes with a smoothly contoured appearance and producing a 2-fold reduction of the DNA length. Protein p5 also binds to ssDNA in the phi 29 replicative intermediates produced in vitro, which are similar in structure to those observed in vivo. Our results strongly suggest that phi 29 protein p5 is the phi 29 SSB protein active during phi 29 DNA replication.     

Polymerization of bacteriophage phi 29 replication protein p1 into protofilament sheets.

Protein p1 (85 amino acids) of the Bacillus subtilis phage phi29 is a membrane-associated protein required for in vivo viral DNA replication. In the present study, we have constructed two fusion proteins, maltose-binding protein (MalE)-p1 and MalE-p1DeltaN33. By using both sedimentation assays and negative-stain electron microscopy analysis, we demonstrated that MalE-p1 molecules self-associated into long filamentous structures, which did not assemble further into larger arrays. These structures were constituted by a core of protein p1 surrounded by MalE subunits. After removal of the MalE component by cleavage with protease factor Xa, the resulting protein p1 filaments tended to associate, forming bundles. The MalE-p1DeltaN33 fusion protein, however, did not self-interact in solution. Nevertheless, after being separated from the MalE domain by factor Xa digestion, protein p1DeltaN33 assembled into long protofilaments that associated in a highly ordered, parallel array forming large two-dimensional sheets. These structures resemble eukaryotic tubulin and bacterial FtsZ polymers. In addition, we show that protein p1 influences the rate of in vivo phi29 DNA synthesis in a temperature-dependent manner. We propose that protein p1 is a component of a viral-encoded structure that associates with the bacterial membrane. This structure would provide an anchoring site for the viral DNA replication machinery.     

Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7

We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.