© 1991 Oxford University Press Nucleic Acids Research, Vol. 19, No. 18 4967-4973 Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity P.Mattila, J.Korpela, T.Tenkanen and K.Pitkanen Finnzymes Oy, Riihitontuntie 14 B, PO Box 148, SF 02200 Espoo, Finland Received June 6, 1991; Revised and Accepted August 22, 1991 ABSTRACT We demonstrate that the DNA polymerase isolated from Our interest was to examine the fidelity of thermostable DNA Downloaded from Thermococcus litoralis (Vent™ DNA polymerase) is polymerases isolated from other sources than the Thermus species the first thermostable DNA polymerase reported having (Thermits aquaticus, Thermus thermophilus, Thermusflavis). That a 3' —5' proofreading exonuclease activity. This is why we decided to study the fidelity of DNA synthesis by the facilitates a highly accurate DNA synthesis In vitro by Vent™ DNA polymerase isolated from Thermococcus litoralis, an extremely thermophilic marine archaebacterium (6). This the polymerase. Mutatlonal frequencies observed in the http://nar.oxfordjournals.org/ base substitution fidelity assays were in the range of organism originates from a submarine thermal vent and can grow 30x10~6. These values were 5-10 times lower at temperatures up to 98 °C. The purified polymerase from this compared to other thermostable DNA polymerases archaebacterium reflects thus high level of thermostability, with lacking the proofreading activity. All classes of DNA a half life of two hours at 100°C. polymerase errors (transitions, transversions, In this study we used three different forms of DNA polymerases frameshift mutations) were assayed using the forward originating from Thermococcus litoralis: a) a native DNA mutational assay (1). The mutation frequencies of polymerase purified directly from Thermococcus litoralis cells Thermococcus litoralis DNA polymerase varied (Vent™N); b) a recombinant form of Vent™ DNA polymerase between 15-35x10-* being 2-4 times lower than equal to native enzyme (Vent™^; and c) a recombinant form by guest on September 27, 2016 the respective values obtained using enzymes without of Vent™ DNA polymerase lacking the 3' —5' exonuclease proofreading activity. We also noticed that the fidelity activity (Vent™,^^). Vent™ polymerases were purified to of the DNA polymerase from Thermococcus litoralis near homogeneity by standard ion exchange chromatography (7). responds to changes in dNTP concentration, units of Cloning and expression of recombinant forms of Vent DNA enzyme used per one reaction and the concentration polymerase was carried out as described (8). As reference of MgSO4 relative to the total concentration of dNTPs enzymes we used Replinase™ (isolated from Thermus flavis) present in the reaction. The high fidelity DNA synthesis and T7 DNA polymerase enzymes. In vitro by Thermococcus litoralis DNA polymerase DNA synthesized by thermostable polymerases at high provides good possibilities for maintaining the genetic temperatures with concomitant higher rates of synthesis and information of original target DNA sequences intact in decreased stability of the DNA helix makes data on the fidelity the DNA amplification applications. of this synthesis essential. If higher fidelity of DNA synthesis can be achieved by a thermostable DNA polymerase than those obtained by Taq DNA polymerase, the use of PCR for a variety of analyses in molecular biology can become even more attractive INTRODUCTION than it is now. The increased number of applications utilizing polymerase chain reaction (PCR) (2,3) has generated increasing demands for MATERIALS AND METHODS thermostable DNA polymerases. Depending on the anticipated use of synthesized DNA, the fidelity of DNA synthesis by the Materials polymerases can be of significant importance. Specifically, The following reagents were kindly supplied by New England cloning and sequencing of cloned DNA utilizing amplified DNA Biolabs: Vent™N DNA Polymerase (254S, Lot 2), Vent™R and generated by PCR requires information about the mutation rates Vent™RE*o- DNA polymerases, T7 DNA polymerase (256S, created by the thermostable enzymes themselves. The fidelity form n, unmodified), Acetylated BSA, necessary restriction of Taq DNA polymerase has already been widely studied (4,5). enzymes for the construction of the DNA substrates needed in These studies indicate that Taq DNA polymerase lacks the 3' —5' the assays for fidelities (Ava n, Kpn I, Pvu I, Pvu II), buffers exonuclease activity and creates both single base substitution for the enzymes previously mentioned. M13mp2A89, errors and transitions, transversions, frameshifts, and/or deletion M13mp2A103, M13mp2G103 and M13mpwt phages and E. coli mutations during in vitro DNA synthesis (4). strains CSH50 and NR9099 and 3'-end labeled oligonucleotide 4968 Nucleic Acids Research, Vol. 19, No. 18 substrate were kind gifts from Dr. Thomas A. Kunkel at NIEHS position 103 are used to construct a gapped heteroduplex (NC, USA). Replinase™ Got WFP1070) was purchased from molecule. This molecule contains a 3'-terminal cytosine residue Du Pont (with buffer); X-gal, IPTG, RNase and Proteinase K in the primer (i.e., minus) strand opposite an adenine residue were purchased from 5' Prime—3' Prime; SeaPlaque GTG and in the template (i.e., plus) strand. Expression of the cytosine- SeaKem LE agaroses were purchased from FMC BioProducts; containing sequence results in a medium-blue plaque phenotype, dNTPs and Sephacryl S-1000 were purchased from Phamacia, whereas expression of the adenine-containing sequence results dGMP (D-9500),Tris (T-1503) and PEG 8000 (P-5413) were in faint-blue plaque phenotype. If the mispaired cytosine is purchased from Sigma; E.coli MC1061 was purchased from the removed prior to extension by the polymerase, subsequent correct culture collection of the Helsinki University, Faculty of incorporation of thymidine opposite the template adenine will Agriculture and Forestry, Department of Microbiology; CaCl2, yield a homoduplex molecule having exclusively a faint-blue MgCl2, MgSO4, NaCl, KH2PO4, Na2HPO4, NH^Cl, Leucine plaque phenotype. However, polymerization to fill the gap and Thiamine-HCl were purchased from Merck; Agar, Yeast without excision of the cytosine will produce a doublestranded Extract and Glucose were purchased from Difco; Tryptone was heteroduplex which, upon transfection, will yield medium-blue purchased from Bio Kar, EDTA and SDS were purchased from plaques ( = 50-60%) and faint-blue plaques (= 40-50%) (4,9). Enprotech and phenol was purchased from Carlo Erba. The proportion of medium- and faint-blue plaques obtained upon BTX ECM 600 electroporation device was used for all the transfection of the reaction products is a function of the extent electroporations. Beckman LS7800 liquid scintillation counter was of terminal mismatch excision. used for terminal mismatch excision assay analysis. Methods 3'—5' exonuclease assay II: Terminal Mismatch Excision by GelDownloaded from Electrophoresis. In the assay the ability of the 3' — 5' exonuclease Preparation of the gapped M13mp2 DNA substrates. M13mp2 activity of a DNA polymerase is used to excise a 3'-terminal base gapped substrates were constructed as earlier described (1,9,10) from a mismatched end of a synthetic oligonucleotide (9). A with minor modifications. The replicative form DNA was purified 20-base oligonucleotide labeled with 32P at the 5'-end was using S-1000 column chromatography the linear flow rate being hybridized to a 27-base template oligonucleotide to create an 14 cm/h (11). To prepare single stranded DNA the phage particles incorrect A • G mispair at the 3'-OH end. Exonuclease assay washttp://nar.oxfordjournals.org/ were first PEG precipitated (1) after which the phage particles performed in 50 /tl reaction containing 50 mM Tris (pH 8.0 at were Proteinase K treated (50 mg/ml Proteinase K with 0.5% 25°C),10 mM MgCl2, 300 ng of 5'-end-labeled DNA (30,000 SDS, 1.5 h at 37°C) and applied to the S-1000 column. Finally cpm//ig) and 0,25 units of Vent™ DNA polymerase. The the pooled fractions were ethanol precipitated. reaction mixture was prewarmed at 37 °C for 3 min before the addition of the enzyme to allow primer annealing. Incubations Transfection of competent cells and plating. A 60 ml aliquot of were for 2, 5, 10, 30 and 60 min at 37°C, respectively, and 5 /tl frozen E.coli MC1061 cells prepared according to the BTX ECM aliquots were taken at the indicated times. (Incubation was carried C 600 operating manual were thawed. 1—1.5 /tl of the reaction out at 37 C in order to avoid the melting of primer from the by guest on September 27, 2016 mixture (1:10 diluted, exept for the reversion assay where template). The reactions were stopped by the addition of EDTA undiluted reaction mixture was used) was added and the mixture to achieve a final concentration of 15 mM. Excision by the DNA was transferred to an ice cold BTX electroporation cuvette P/N polymerase was monitored by gel electrophoresis of 3 /tl aliquots 620 (2 mm gap). A single pulse was delivered at 2.5 kV and in a 20% denaturing polyacrylamide gel at the said time intervals. 129 fl . The resultant pulse lengths varied around 6 ms and the The gel was then dried and used to expose Kodak XAR film. 9 transfection efficiencies varied between 1—6xlO The radioactivity in each band was quantitated by cutting the transfectants//tg DNA. Immediately after the pulse 2 ml of SOC bands from the gel and counting the radioactivity in a liquid (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 10 mM scintillation counter. MgCl2, 20 mM glucose) was added. After thorough mixing the cells were plated with E.coli CSH50 cells (12). Scoring of a-complementation mutants was performed as Reversion Assay for Base Substitution Fidelity. Base substitution described (1,12,13). The DNA polymerase reactions were errors occurring during in vitro DNA synthesis can be monitored prewarmed to 72 °C (for Vent™ DNA polymerases and in the A89 opal codon reversion assay, which .detects eight of Replinase™) or 37 °C (for T7 DNA polymerase) for five nine possible single-base substitution errors at this codon (1,4, minutes prior to the addition of the DNA polymerases.