Replication of bacteriophage phi 29 DNA in vitro: the roles of terminal protein and DNA polymerase.

Abstract

ABSTRACT429 DNA replication is initiated by the formation of a covalent complex between the viral-coded terminal protein and dAMP (TP-dAMP). This initiation reaction system has been reconstituted from two phage-encoded proteins, the terminal protein and DNA polymerase. The 429 DNA polymerase was purified from phage-infected cells by using poly (dA) All of the known initiation events of DNA replication start from small RNA or DNA primers with the exception of bacteriophage 429 and adenovirus and their related viruses.These viral DNAs employ a viral-coded terminal protein as a primer for their DNA replication (1-6). The phage 429 genome is a linear double-stranded DNA of about 18,000 base pairs, whose 5' ends are linked to a terminal protein of molecular mass 30 kDa (DNA-protein) (7). DNA sequence analysis has shown that the 429 genome has six-base-pair inverted repeats at the termini (8, 9).Recently, we described a soluble enzyme system for 429 DNA replication that uses exogeneous 429 DNA-protein isolated from phage particles as template (2, 3). The system has facilitated elucidation of the basic mechanism of 429 DNA replication (2-5, 10-14). The first step in the replication of 429 DNA is the formation of a covalent complex between free terminal protein (TP) and dAMP, the first nucleotide of the 5' end of 429 DNA, in the presence of 429 DNAprotein as template. The cell-free extract can catalyze both TP-dAMP complex formation and DNA replication (initiation and elongation). Both activities were copurified through several steps of column chromatography as an apparent replication complex (4). Furthermore, the TP-dAMP complex has been shown to be elongated by the purified replication complex (4), indicating that TP-dAMP indeed functions as a primer for DNA replication.The soluble enzyme system also facilitated identification of the gene products required for DNA replication. Thus far, two gene products have been shown to be necessary for initiation of 429 DNA replication: gene 3 (4, 10, 11) and gene 2 (10-12). Gene 3 is the structural gene for the TP (15). We recently found that phage 429 induces its own DNA polymerase (12). The polymerase activity isolated from cells infected with a gene 2 temperature-sensitive mutant was thermolabile compared with wild-type polymerase. This indicates that gene 2 is the structural gene for the 429 DNA polymerase (12).In this report, we describe the purification of 429 TP and 429 DNA polymerase and show that these two proteins are sufficient to catalyze the initiation of )29 DNA replication. These results provide further insight into protein-primed initiation of linear nonredundant duplex DNA replication. MATERIALS AND METHODSMaterials. Bacillus subtilis SCR 463 (polA phe hisB trp) was used for preparation of phage-infected cell extracts. Escherichia coli strains RLM866 [C600rj mj thr leu pro (XcI857 ABamHI) supE tonA lac] and RLM 841 [C600rj mrecA-, (XcI'km)/pATY2131 (AmPr PL promoter)] were provided by R. McMacken. Phage 429 wild type, sus2 and sus3 (nonsense mutants in genes...

Replication of phage φ29 DNAin vitro: role of the viral protein p6 in initiation and elongation

“…Replication of 029 is initiated at either DNA end (4)(5)(6) by the covalent attachment of 5'-dAMP to the serine residue 232 (7) of a free molecule of, the terminal protein p3 (8,9). The formation of the p3-dAMP initiation complex is catalyzed by the 029 DNA polymerase p2 (10,11) and requires the presence of a 029 DNA specific DNA template (12,13). Proteins p2 and p3 have been purified (10,11,14) and, when added to the 029 DNA-protein p3 template, the p3-dAMP initiation complex is formed and further elongated to produce full-length 029 DNA (15), indicating that the 029 DNA Nucleic Acids Research polymerase is the only one required for the in vitro synthesis of 029 DNA.…”

“…The formation of the p3-dAMP initiation complex is catalyzed by the 029 DNA polymerase p2 (10,11) and requires the presence of a 029 DNA specific DNA template (12,13). Proteins p2 and p3 have been purified (10,11,14) and, when added to the 029 DNA-protein p3 template, the p3-dAMP initiation complex is formed and further elongated to produce full-length 029 DNA (15), indicating that the 029 DNA Nucleic Acids Research polymerase is the only one required for the in vitro synthesis of 029 DNA. However, under these conditions the rate of elongation is low, suggesting the need of accessory proteins for the efficient replication of 029 DNA.…”

Isolation of suppressors of the temperature-sensitive growth caused by a nonsense mutation in gene 1 of <i>Bacillus subtilis</i> phage ø29 using hydroxylamine

“…To avoid accumulation of numerous mutations Bacillus subtilis phage ø29 has a linear double-stranded DNA genome with a terminal protein covalently linked to each 5′ end (Hirokawa, 1972;Ortín et al, 1971;Yanofsky et al, 1976;, and has been studied extensively as a model system of the protein-priming DNA replication in which a viral-encoded protein provides the hydroxyl group needed by viral DNA polymerase to initiate the DNA synthesis (Matsumoto et al, 1983(Matsumoto et al, , 1984Peñalva and Salas, 1982;Watabe et al, 1982Watabe et al, , 1984. The detailed mechanism and proteins involved in the reaction has been clarified in vitro (Blanco et al, 1986(Blanco et al, , 1992(Blanco et al, , 1994Gutiérrez et al, 1991).…”

“…Although several characteristics of gp1 have been reported Salas, 1997, 1998;Bravo et al, 2000;Takeuchi et al, 1998), its role in viral DNA replication is still unclear. By isolating temperature-resistant revertants with spontaneous mutations from ø29 gene 1 mutants, we have identified several second-site mutations that suppressed the defect of the gene 1 mutations at 42°C (Tone et al, 2012a); these suppressor mutations were located in gene 3 and gene 5. ø29 gene 3 product (gp3) is a key protein for ø29 DNA replication: it acts as a protein primer for the initiation of DNA replication and is also the component of the replication origin as a terminal protein (Matsumoto et al, 1983(Matsumoto et al, , 1984Peñalva and Salas, 1982;Watabe et al, 1982Watabe et al, , 1984. On the other hand, ø29 gene 5 product (gp5) is a single-stranded DNA binding (SSB) protein required for viral DNA replication in a growth-temperature dependent fashion (Martín et al, 1989; in the ø29 genomes, ø29 ns1 was incubated with 0.2 M hydroxylamine at 37°C for 20 hours to give a survival of 10 -2 ; on average, one mutation per genome is introduced in this condition (Matsumoto et al, 1986).…”

The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization.

“…The fact that the YGDTDS sequence is not restricted to RNA-or DNA-primed DNA polymerases but is also present in DNA polymerases for terminal protein-containing DNA genomes is of additional interest because it was not known whether the specific protein-priming activity of these DNA polymerases had its own catalytic site or shared general domains of the polymerization active site. Bacteriophage 429 DNA polymerase, product of viral gene 2 (Mr, 66,520), initiates replication (24)(25)(26) by catalyzing the formation of a phosphoester bond between the 8-OH group of Ser-232 in the terminal protein (p3) and 5'-dAMP, the 5' terminal nucleotide at both DNA ends (27). The 429 DNA polymerase also has 5' -* 3' polymerization and 3'-* 5' exonuclease activities characteristic of other DNA replicases.…”