A 3′ to 5′ exonuclease activity is associated with phage Ø29 DNA polymerase

Watabe, Kounosuke; Leusch, Mark S.; Ito, Junetsu

doi:10.1016/s0006-291x(84)80235-1

Cited by 19 publications

(5 citation statements)

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“…The products of genes S and 6 were shown to be involved in elongation in vivo (31), and the fact that extracts from susl7-infected B. subtilis can form the p3-dAMP initiation complex in vitro (10, 11) might suggest that the gene 17 product is also involved in elongation. The gene 6 product has been recently purified and shown to stimulate the initiation reaction in vitro (32).…”

Section: Discussionmentioning

confidence: 99%

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

Blanco¹,

Salas²

1985

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Discussionmentioning

confidence: 99%

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

Blanco¹,

Salas²

1985

Proc. Natl. Acad. Sci. U.S.A.

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“…Use of single-stranded M13 DNA as template gave a rolling-circle mode of replication, in which the polymerase repeatedly copied around the circular template via its strand displacement activity, yielding a product of concatenated M13 repeats. The DNA polymerase's associated 3 0 -5 0 exonuclease proofreading activity results in a low intrinsic error rate of 10 À6 -10 À7 (Watabe et al, 1984;Blanco and Salas, 1985) and the accumulation of mutations in MDA products at a Multiple displacement amplification and microbial ecology EK Binga et al rate of only 10 À5 -10 À6 (Esteban et al, 1993;Nelson et al, 2002), nearly 1000-fold less than for PCR using the Taq DNA polymerase (Dunning et al, 1988;Saiki et al, 1988). The first use of MDA for amplifying whole genomes targeted human DNA (Dean et al, 2002) and as few as 90 copies of the genome (0.3 ng DNA) yielded more than 30 mg of product.…”

Section: A Brief History Of Mdamentioning

confidence: 99%

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

2008

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Microbial ecology is a field that applies molecular techniques to analyze genes and communities associated with a plethora of unique environments on this planet. In the past, low biomass and the predominance of a few abundant community members have impeded the application of techniques such as PCR, microarray analysis and metagenomics to complex microbial populations. In the absence of suitable cultivation methods, it was not possible to obtain DNA samples from individual microorganisms. Recently, a method called multiple displacement amplification (MDA) has been used to circumvent these limitations by amplifying DNA from microbial communities in low-biomass environments, individual cells from uncultivated microbial species and active organisms obtained through stable isotope probing incubations. This review describes the development and applications of MDA, discusses its strengths and limitations and highlights the impact of MDA on the field of microbial ecology. Whole genome amplification via MDA has increased access to the genomic DNA of uncultivated microorganisms and low-biomass environments and represents a 'power tool' in the molecular toolbox of microbial ecologists.

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“…In contrast to the homology to the prokaryotic q~29 polymerase, which may reflect convergent evolution of sequences with special properties, the homologies to the viral enzymes are taken to represent evolutionary conservation, whereby the VZV polymerase exhibits the greatest, and the adenovirus the least evolutionary relatedness to the HSV sequence. Interestingly, except for VZV for which no data are available, all of these enzymes possess an associated nuclease activity (Knopf, 1979;Chalberg & Englund, 1979;Kallin et al, 1985;Field et al, 1984;Watabe et al, 1984). Another interesting feature is the conserved positioning of aspartic acid and tyrosine residues, separated by four amino acids, with the alanine to valine substitution site between these two residues in a region of apparent lower homology.…”

Section: Short Communicationmentioning

confidence: 99%

The Herpes Simplex Virus Type 1 DNA Polymerase Gene: Site of Phosphonoacetic Acid Resistance Mutation in Strain Angelotti is Highly Conserved

Knopf

1987

Journal of General Virology

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SUMMARYBy comparative sequence analysis of the herpes simplex virus type 1 DNA polymerase gene of strain Angelotti and a phosphonoacetic acid-resistant (PAA9 derivative, the site of the PAA r mutation was identified as a single nucleotide (C --, T) conversion within the mapping limits of the known PAA r mutations of strains KOS and 17. The conservative amino acid change at residue 719 from alanine to valine results in a radical change in the properties of the polymerase, rendering the mutant enzyme resistant to PAA and various antiviral compounds. Amino acid homologies as well as secondary structure analysis reveal that the PAA ~ mutation is contained in a 14 amino acid sequence which is highly conserved, and detected in the central domain of prokaryotic and eukaryotic DNA polymerases.The herpes simplex virus (HSV) DNA polymerase represents one of the major target enzymes of modern antiviral chemotherapy. The unique response of the enzyme towards antiherpetic drugs, and the ease by which drug-resistant variants with altered enzymic properties are isolated, have rendered the HSV polymerase gene an excellent model system for correlating genetic changes with altered enzymic functions. Biochemical studies on the polymerase activity of HSV type 1 (HSV-1) and type 2 variants and intertypic recombinants reveal that mutations causing resistance to the pyrophosphate analogue phosphonoacetic acid (PAA) also render the enzyme resistant to nucleoside analogues (Furman et al., 1981 ;Knopf et al., 1981 ;Coen et al., 1982). The mutually exclusive inhibition of the wild-type enzyme by pyrophosphate and nucleoside analogues suggests that the binding sites for dNTP and PP~ are kinetically overlapping (Frank & Cheng, 1985). By marker rescue studies (Chartrand et al., 1979;Coen et al., 1984) and sequence analysis (Gibbs et al., 1985), the limits for the PAA resistance (PAA r) mutations of strains KOS and 17 were narrowed to a region between amino acid residues 535 and 924. Owing to the extended homologies noted between the predicted DNA polymerase proteins of herpesviruses, poxviruses and adenoviruses (Quinn & McGeoch, 1985;Gibbs et al., 1985;Earl et al., 1986), this C terminal domain has been proposed to participate in the organization of the catalytic site of the enzyme.In the absence of crystallographic data, one way to obtain information on the structure of the catalytic site is to determine the sequence changes in mutants that alter the enzyme's behaviour towards substrate and substrate analogues. In this report, we present an analysis of the differences in structure and function of the DNA polymerase genes of wild-type HSV-1 strain Angelotti (ANG) and of a PAA ~ variant. The latter virus was selected for resistance to PAA by sequential passages ofHSV-1 ANG in Rita cells (RC-37) at a PAA concentration of 300 ~tg/ml, and was plaque-purified three times. This PAA r variant (isolate number III-2) was equally resistant to PAA (e.o.p. + 100 lag/ml PAA, 1.04 + 0-2) and acyclovir (ACV; e.o.p. + 50 ~tM-ACV, 1.0 + 0.15), and otherwise ...

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A 3′ to 5′ exonuclease activity is associated with phage Ø29 DNA polymerase

Cited by 19 publications

References 6 publications

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

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

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

The Herpes Simplex Virus Type 1 DNA Polymerase Gene: Site of Phosphonoacetic Acid Resistance Mutation in Strain Angelotti is Highly Conserved

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