In vitro complex formation between bacteriophage φ29 terminal protein and deoxynucleotide

It has previously been shown that the 5′-terminal deoxyadenosine residue of each φ29 DNA strand is linked covalently to the 30,000 dalton terminal protein. When extracts prepared from φ29-infected$\text{Bacillus}\text{subtilis}$ cells are incubated with [α-³²p]dATP, complexes consisting of the nucleotide covalently linked to a 30,000 dalton protein can be detected. The formation of this complex requires the presence of φ29 DNA containing the bound 30,000 dalton terminal protein and Mg⁺⁺. When uninfected cell extracts were used, there was no complex formation. When [α-³²p]dCTP was used in place of [α-³²p]dATP, no complex was formed. DNA-protein templates prepared from φ29 related phages, φ15, Nf, M2Y and GA-1, also supported the complex formation in various degrees. These results support the hypothesis that the terminal protein serves as a primer for the initiation of φ29 DNA replication.     

Bacteriophage phi29 terminal protein: its association with the 5' termini of the phi29 genome.

The location of the protein bound to bacteriophage phi29 DNA has been studied with restriction endonucleases, exonucleases, and polynucleotide kinase. The protein is invariably associated with the two terminal DNA fragments generated by restriction endonucleases. The phi29 DNA prepared with or without proteinase K treatment is resistant to the action of the 5'-terminal-specific exonucleases, lambda-exonuclease and T7 exonuclease. The phi29 DNA is also inaccessible to phosphorylation by polynucleotide kinase even after treatment with alkaline phosphatase. On the other hand, phi29 DNA is sensitive to exonuclease III, and the 3' termini of the DNA can be labeled by incubating with alpha-[32P]ATP and terminal deoxynucleotidyl transferase. The protein remains associated with the phi29 DNA after treatment with various chaotropic agents, including 8 M urea, 6 M guanidine-hydrochloride, 4 M sodium perchlorate, 2 M sodium thiocyanate, and 2 M LiCl. These results are consistent with the notion that the protein is linked covalently to the 5' termini of the phi29 DNA.     

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.     

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.     

Formation of a covalent complex between the terminal protein of pneumococcal bacteriophage Cp-1 and 5''-dAMP.

Incubation of extracts of Cp-1-infected Streptococcus pneumoniae with [alpha-32P]dATP produced a labeled treatment with micrococcal nuclease and sensitive to treatment with proteinase K. Incubation of the 32P-labeled protein with 5 M piperidine for 4 h at 50 degrees C released 5'-dAMP, indicating that a covalent complex between the terminal protein and 5'-dAMP was formed in vitro. When the four deoxynucleoside triphosphates were included in the reaction mixture, a labeled complex of slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels than the terminal protein-dAMP complex was also found, indicating that the Cp-1 terminal protein-dAMP complex can be elongated and, therefore, that it is an initiation complex. Treatment of the 32P-labeled terminal protein-dAMP complex with 5.8 M HCl at 110 degrees C for 2 h yielded phosphothreonine. These results, together with the resistance of the terminal protein-DNA linkage to hydroxylamine, suggest that the Cp-1 terminal protein is covalently linked to the DNA through a phosphoester bond between L-threonine and 5'-dAMP, namely, a O-5'-deoxyadenylyl-L-threonine bond.     

Initiation of adenovirus DNA replication: detection of covalent complexes between nucleotide and the 80-kilodalton terminal protein

We have previously shown that the 5'-terminal deoxycytidine residue of each nascent adenovirus 5 DNA strand synthesized in vitro is covalently linked to the 80-kilodalton (kd) terminal protein precursor via a phosphodiester bond to a serine residue in the protein. When extracts prepared from adenovirus 5-infected cells are incubated with [alpha-33P]dCTP as the only added deoxynucleoside triphosphate, complexes consisting of nucleotide covalently linked to the 80-kd protein can be detected. The nucleotide moieties present in such complexes include d(pC) and d(pCpA), the 5'-terminal nucleotide and dinucleotide of adenovirus 5 DNA, respectively, as well as some longer oligonucleotides. The formation of these complexes requires the presence of adenovirus DNA containing the attached 55-kd terminal protein and ATP. Extracts from H5ts125-infected cells which are defective in DNA replication catalyze complex formation to the same extent as extracts prepared from wild-type infected cells; thus, the presence of the adenovirus-coded 72-kd DNA-binding protein is apparently not required. Most, if not all, of the 80-kd protein-nucleotide complexes that are formed are noncovalently bound to the input viral DNA. These observations are consistent with the protein-priming model for the initiation of adenovirus DNA replication.     

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.     

Initiation of phage phi 29 DNA replication by mutants with deletions at the amino end of the terminal protein

Series of deletions at the amino end of protein p3, the phage phi 29 DNA terminal protein (TP), have been constructed and characterized. Measurements of the activity of the deletion mutants in the formation of the protein p3-dAMP initiation complex in vitro indicate the dispensability of the first 13 amino acids (aa) of the protein. The activity of protein p3 decreased considerably when 17 or more aa were deleted. The results on the in vitro phi 29 DNA replication primed by the p3 deletion mutants correlated very well with those obtained in the formation of the TP-dAMP initiation complex.     

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. I. Characterization of the termination and reinitiation reactions

The phi X174 (phi X) gene A protein-mediated termination and reinitiation of single-stranded circular (SS(c] phi X viral DNA synthesis in vitro were directly and independently analyzed. Following incubation together with purified DNA replication enzymes from Escherichia coli, ATP, [alpha-32P]dNTPs, and either the phi X A protein and phi X replicative form I (RF I) DNA, or the purified RF II X A complex, the phi X A protein was detected covalently linked to newly synthesized 32P-labeled DNA. Formation of the phi X A protein-[32P]DNA covalent complex required all the factors necessary for phi X (+) SS(c) DNA synthesis in vitro. Thus, it was a product of the reinitiation reaction and an intermediate of the replication cycle. Identification of this complex provided direct evidence that reinitiation of phi X (+) strand DNA synthesis involved regeneration of the RF II X A complex. Substitution of 2',3'-dideoxyguanosine triphosphate (ddGTP) for dGTP in reaction mixtures resulted in the formation of covalent phi X A protein 32P-oligonucleotide complexes; these complexes were trapped analogues of the regenerated RF II X A complex. They could not act catalytically due to the presence of ddGMP residues at the 3'-termini of the oligonucleotide moieties. Reaction mixtures containing ddGTP also yielded nonradioactive (+) SS(c) DNA products derived from circularization of the displaced (+) strand of the input parental template DNA. The formation of the phi X A protein-32P-oligonucleotide complexes and nonradioactive (+) SS(c) DNA were used to assay both reinitiation and termination reactions, respectively. Both reactions required DNA synthesis from the 3'-hydroxyl primer at nucleotide residue 4305 which was formed by cleavage of phi X RF I DNA by the phi X A protein. Elongation of this primer by 18, but not 11 nucleotides was sufficient to support each reaction. Reinitiation reactions proceeded rapidly and were essentially complete after 90 s. In contrast, when ddGTP was replaced with dGTP in reaction mixtures, DNA synthesis proceeded with linear kinetics for up to 10 min. These results suggested that in the presence of all four dNTPs, active templates supported more than 40 rounds of DNA synthesis.     

Synthesis of phage-specific transfer RNA molecules by vibriophage phi 149

32P-Labelled tRNA was isolated from uninfected and phage phi 149-infected Vibrio cholerae cells. These tRNA preparations were then hybridised with DNA isolated from phage phi 149. Significant hybridisation was observed only with tRNA from phage phi 149-infected cells. This strongly suggests that infection of classical vibrio with phage phi 149 results in the synthesis of phage-specific tRNA molecules.     

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.     

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. II. Structural characterization of the covalent phi X A protein-DNA complex

In the preceeding paper (Brown, D. R., Roth, M. J., Reinberg, D., and Hurwitz, J. (1984) J. Biol. Chem. 259, 10545-10555), it was shown that following bacteriophage phi X174 (phi X) DNA synthesis in vitro using purified proteins, the phi X A protein could be detected covalently linked to nascent 32P-labeled DNA. This phi X A protein-[32P]DNA complex was the product of the reinitiation reaction. The phi X A protein-[32P]DNA complex could be trapped as a protein-32P-oligonucleotide complex by the inclusion of ddGTP in reaction mixtures. In this report, the structure of the phi X A protein-32P-oligonucleotide complex has been analyzed. The DNA sequence of the oligonucleotide bound to the phi X A protein has been determined and shown to be homologous to the phi X (+) strand sequence immediately adjacent (3') to the replication origin. The phi X A protein was directly linked to the 5' position of a dAMP residue of the oligonucleotide; this residue corresponded to position 4306 of the phi X DNA sequence. The phi X A protein-32P-oligonucleotide complex was exhaustively digested with either trypsin or proteinase K and the 32P-labeled proteolytic fragments were analyzed. Each protease yielded two different 32P-labeled peptides in approximately equimolar ratios. The two 32P-labeled peptides formed after digestion with trypsin (designated T1 and T2) and with proteinase K (designated PK1 and PK2) were isolated and characterized. Digestion of peptide T1 with proteinase K yielded a product which co-migrated with peptide PK2. In contrast, peptide T2 was unaffected by digestion with proteinase K. These results suggest that the phi X A protein contains two active sites that are each capable of binding covalently to DNA. The peptide-mononucleotide complexes T1-[32P]pdA and T2-[32P]pdA were isolated and subjected to acid hydrolysis in 6.0 N HCl. In each case, the major 32P-labeled products were identified as [32P] phosphotyrosine and [32P]Pi. This indicates that each active site of the phi X A protein participates in a phosphodiester linkage between a tyrosyl moiety of the protein and the 5' position of dAMP.     

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.     

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.     

Initiation of phage phi 29 DNA replication by mutants with deletions at the carboxyl end of the terminal protein

Series of deletions at the C end of p3, the phage phi 29 DNA terminal protein (TP), have been constructed and characterized. Measurements of the activity of those deletion mutants in the formation of the p3-dAMP initiation complex in vitro indicate the need of an intact C-end for the normal TP primer function in DNA replication. It appears that the region at the C-end between aa 240 and 262 of p3, or part of it, might be essential for the normal TP function.     

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.     

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.