Replication of phage phi 29 DNA with purified terminal protein and DNA polymerase: synthesis of full-length phi 29 DNA.

Abstract

A system that replicates bacteriophage 429DNA with protein p3 covalently attached to the two 5' ends, using as the only proteins the 4)29 DNA polymerase and the terminal protein, is described. Restriction analysis of the 32P-labeled DNA synthesized in vitro showed that all 429 DNA fragments were labeled. Analysis by alkaline sucrose gradient centrifugation of the DNA labeled during a 10-min pulse showed that, after a 20-min chase, about half of the DNA MATERIALS AND METHODSAssay for Formation of the p3-dAMP Initiation Complex. The incubation mixture contained, in 0.05 ml, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol, 1 mM spermidine, 0.25 ,uM [a-32P]dATP (5 ,uCi; 1 Ci = 37 GBq), 1 ,ug of 429 DNA-protein p3, 300 ng of purified protein p3 (19) obtained from I. Prieto, 20 ng of purified protein p2 (15) and, when indicated, 3 ,ug of a fraction of uninfected B. subtilis cells prepared as described by Blanco and Salas (15) as a source of host factor(s). After incubation for 20 min at 30°C, the samples were filtered through Sephadex G-50 spuncolumns (20) and processed as described by Pefialva and Salas (12).Replication Assay With Phage 429 DNA-Protein p3 as Template. The incubation mixture was as described for the initiation reaction except that it contained 10 ,uM [a-32P]dATP (2 ,Ci); 20 ,uM each dGTP, dCTP, and dTTP (P-L Biochemicals), 1 mM ATP; 5% (vol/vol) glycerol; and bovine serum albumin (0.1 mg/ml). Besides purified proteins p2 and p3, the components indicated in each case were added. After 20 min at 30°C, the reaction was stopped by adding 10 mM EDTA/0.1% NaDodSO4 and heating for 10 min at 68°C, and the samples were filtered through Sephadex G-50 spuncolumns as described (20) Then,ug) was added to stop further initiations and, at the indicated times, dATP up to 10 ,uM was added as well as 20 ,uM ofdCTP, dTTP, and dGTP. The incubation was continued at 30°C with the additions indicated in each case. At different times, samples were removed, the reaction was stopped by addition of 10 mM EDTA/0.1% NaDodSO4 and heating for 10 min at 68°C, and the labeled DNA was subjected to restriction analysis as described below.Restriction Analysis of the Replicated 429 DNA. The DNA labeled in the replication and elongation assays described above with phage 029 DNA-protein p3 as template was treated with proteinase K (200 ,ug/ml) in the presence of0.5% NaDodSO4 during 5 hr at 37°C. The samples were filtered through Sephadex G-50 spun-columns, and the excluded fractions were treated with phenol/chloroform as described (20), precipitated with ethanol, and treated with HindIll. The fragments were separated in a 3.5% polyacrylamide slab gel. After electrophoresis, the gel was dried and autoradiographed with intensifying screens at -70°C. Quantitation was 6404The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Genetic analysis of bacteriophage N4 adsorption

“…Another example of polypeptide injection occurs during infection with the Bacillus subtilis bacteriophage 4)29. A 28-kDa polypeptide (terminal protein) is covalently attached to the double-stranded-DNA 4)29 genome at each 5' terminus (33) and participates in a priming mechanism for DNA replication (2). Terminal protein differs from N4 virion RNA polymerase in its relatively small size and its covalent attachment to the +29 DNA.…”

φ29 Family of Phages

“…After the initiation, sliding-back, and transition steps, continuous polymerization, carried out by a single 29 DNA polymerase molecule, completes the replication of the almost 20-kb DNA strand (30). Using primed M13 DNA as the template, the 29 DNA polymerase is able to synthesize DNA chains of more than 70 kb (25).…”

“…The DNA polymerase of 29 has been analyzed in detail (for reviews, see references 31 and 32). The monomeric 29 DNA polymerase, which has a size of only about 66 kDa, catalyzes both the initiation and elongation stages of DNA synthesis (29,30). To accomplish this, it is able to carry out two distinguishable synthetic reactions: TP deoxynucleotidylation and DNA polymerization.…”

The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein

“…The 29-encoded TP and the DNA polymerase are the only two proteins required in a minimal in vitro 29 DNA replication system (29). Contrary to the 29 DNA amplification system, the minimal replication system requires high concentrations of template TP-DNA and is limited to one or two rounds of 29 DNA replication.…”