A novel DNA polymerase induced by Bacillus subtilis phage phi 29.

A novel DNA polymerase induced by Bacillus subtilis bacteriophage phi 29 has been identified. This polymerase can be separated from host DNA polymerase, by fractionation of extracts prepared from phage infected cells, using phosphocellulose chromatography. The isolated polymerase prefers poly(dA)oligo(dT) as template. The DNA polymerase isolated from the cells infected with a gene 2 temperature sensitive mutant (ts2) showed greater heat-lability than that induced by wild type phi 29. The ts2 DNA polymerase was also thermolabile for its activity in the formation of a covalent complex between phi 29 terminal protein and dAMP, the initiation step of phi 29 DNA replication. These findings indicate that gene 2 is the structural gene for a phi 29 DNA polymerase required for the complex formation step of DNA initiation.     

X-ray scattering of the non-isometric Bacillus subtilis phage phi29.

The non-isometric virus φ29 and its empty capsid have enough elements of symmetry so that their size and approximate shape can be determined by low-angle X-ray scattering. The scattering curve of the virus can be simulated by a cylinder of 390 Å diameter and 460 Å height. The protein coat has a thickness of about 30 Å. The DNA is packed tightly and regularly in the phage head.     

Overproduction and purification of the connector protein of Bacillus subtilis phage phi 29.

A phi 29 DNA fragment containing genes 10 and 11, coding for the connector protein and the lower collar protein, respectively, has been cloned in the pBR322 derivative plasmid pKC30 under the control of the PL promoter of phage lambda. Two polypeptides with the electrophoretic mobility of proteins p10 and p11 were labelled with 35S-methionine after heat induction. The proteins were characterized as p10 and p11 by radioimmunoassay and they represented about 10% and 7%, respectively, of the total E. coli protein after 4 hours of induction. These proteins represent less than 1% of the B. subtilis protein in phi 29-infected cells. Protein p10 has been highly purified from the E. coli cells carrying the recombinant plasmid. Antibodies raised against the purified protein p10 reacted with the connector protein produced in phi 29-infected B. subtilis.     

Characterization and cloning of gene 5 of Bacillus subtilis phage phi 29

Sequencing of the phi 29 DNA region [open reading frames (ORFs) 12, 11 and 10] between genes 6 and 4 of the mutant ts5(219) showed that a G in the wild-type phage had been changed to an A in the mutant at position 218 of ORF 10 indicating that this ORF corresponds to gene 5. ORF 10 was cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda and, after heat induction of the Escherichia coli cells carrying the recombinant plasmid pGM26, a 12-kDa protein was overproduced, accounting for about 5% of the de novo synthesized protein. Introduction of a nonsense mutation in ORF 10 indicated that the latter codes for the 12-kDa protein. The predicted secondary structure, the hydrophilicity values and the antigenic regions of protein p5 are discussed.     

Implication of the prohead RNA in phage phi29 DNA packaging

RNA is an important component of many biological processes, including DNA encapsidation of bacteriophage phi29 of Bacillus subtilis. Interestingly, the prohead RNA is involved in this encapsidation, and was found in monomer, dimer, pentamer and hexamer conformations. This article presents and debates current knowledge about the prohead RNA structures, mechanisms, and roles in DNA encapsidation. A new dimer structure is presented, and its specific role in DNA encapsidation is discussed.     

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.     

Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29.

We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.     

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.     

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.     

Overproduction and purification of protein P6 of Bacillus subtilis phage phi 29: role in the initiation of DNA replication.

A phi 29 DNA fragment containing gene 6, required for DNA replication, has been cloned in plasmid pPLc28 under the control of the PL promoter of phage lambda. A polypeptide with an electrophoretic mobility close to that of p6 was labelled with 35S-methionine after heat induction. This protein, representing about 4% of the total E. coli protein after 1 h of induction, was obtained in a highly purified form. The protein was characterized as p6 by amino acid analysis and NH2-and COOH-terminal sequence determination. Protein p6 has an apparent molecular weight of 23,600, suggesting that the native form of the protein is a dimer. The purified protein p6 stimulated the protein-primed initiation of phi 29 DNA replication when added to purified proteins p2 (phi 29-coded DNA polymerase) and p3 (terminal protein).     

Relevance of UP elements for three strong Bacillus subtilis phage phi29 promoters

Various Escherichia coli promoters contain, in addition to the classical -35 and -10 hexamers, a third recognition element, named the UP element. Located upstream of the -35 box, UP elements stimulate promoter activity by forming a docking site for the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Accumulating genetic, biochemical and structural information has provided a detailed picture on the molecular mechanism underlying UP element-dependent promoter stimulation in E.coli. However, far less is known about functional UP elements of Bacillus subtilis promoters. Here we analyse the strong early sigma(A)-RNA polymerase-dependent promoters C2, A2c and A2b of the lytic B.subtilis phage phi29. We demonstrate that the phage promoters contain functional UP elements although their contribution to promoter strength is very different. Moreover, we show that the UP element of the A2b promoter, being critical for its activity, is located further upstream of the -35 box than most E.coli UP elements. The importance of the UP elements for the phage promoters and how they relate to other UP elements are discussed.     

Characterization of a protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi29.

We have isolated a covalent DNA-protein complex from bacteriophage φ29 particles. Polyacrylamide gel electrophoresis and tryptic peptide analysis showed that the protein present in the complex is very similar or identical to p3, an early induced protein essential for viral DNA replication.When the DNA-protein complex is treated with the restriction endonuclease EcoRI, the protein is specifically associated to the two terminal fragments, A and C. The protein is probably linked to the 5′ termini of the DNA since proteinase K-treated DNA is resistant to phosphorylation with polynucleotide kinase, even after treatment with alkaline phosphatase, while it is sensitive to exonuclease III. By electron microscopy the protein is visualized as a dot located at the ends of unit length DNA molecules.Mixed infection of Bacillus subtilis, at 42 °C, with ts² mutants in cistrons 2 and 3 only produces ts 2 progeny. This finding suggests that an inactive protein p3 bound to the DNA of the ts 3 mutant is not replaced by a functional protein and, as a consequence, replication of the ts 3 DNA does not occur.