Creation of genetic information by DNA polymerase of the thermophilic bacterium Thermus thermophilus.

Genetic information encoded in a template of a genome is replicated in a complementary way by DNA polymerase or RNA polymerase with high fidelity; no creation of information occurs in this reaction unless an error occurs. We report here that DNA polymerase of the thermophilic bacterium Thermus thermophilus can synthesize up to 200 kb linear double-stranded DNA in vitro in the complete absence of added primer and template DNAs, indicating that genetic information is actively created by protein. This ab initio DNA synthesis occurs at 74 degrees C and requires magnesium ion. There is a lag time of approximately 1 h and then the reaction proceeds linearly. The synthesized DNAs have a variety of sequences; they are mostly tandem repetitive sequences, e.g. (CATGTATA) n , (TGTATGTATACATACATA) n and (TATACGTA) n . Some degenerate sequences of these basic repeat units are also found. The similar repetitive sequences are found in many natural genes. These results, together with similar results found using DNA polymerase of archaeon Thermococcus litoralis , suggest that creative, non-replicative synthesis of DNA by protein was a driving force for diversification of genetic information at a certain stage of the evolution of life on the early earth.     

Elongation of tandem repetitive DNA by the DNA polymerase of the hyperthermophilic archaeon Thermococcus litoralis at a hairpin-coil transitional state: a model of amplification of a primordial simple DNA sequence

DNA is replicated by DNA polymerase semiconservatively in many organisms. Accordingly, the replicated DNA does not become larger than the original DNA (template DNA), implying that replicative synthesis by DNA polymerase alone cannot explain the diversification of primordial simple DNA. We demonstrate that a single-stranded tandem repetitive oligodeoxyribonucleic acid (oligoDNA) composed of a palindromic or quasi-palindromic motif sequence and 25-50% GC content is elongated in vitro to more than 20,000 bases at 70-74 degrees C by the DNA polymerase of the hyperthermophilic archaeon Thermococcus litoralis without a bimolecular primer-template complex. The efficiency of elongation decreased when the palindromic structure of the oligoDNA was destroyed or when the GC content of the oligoDNA was outside the range of 25-50%. The thermal melting transition profile of the oligoDNA, as observed by ultraviolet spectroscopy, exhibited a biphasic curve, reflecting a duplex-hairpin transition at 31-40 degrees C and a hairpin-coil transition at 70-77 degrees C. The optimal reaction temperature for the elongation, for instance, of oligoDNA (AGATATCT)(6) (72 degrees C) was very close to its hairpin-coil transition melting temperature (70.4 degrees C), but was markedly higher than the temperature at which duplex oligoDNA can exist stably (<35.9 degrees C). These results suggest that a hairpin-based "intramolecular primer-template structure" is formed transiently in the oligoDNA, and it is elongated by the DNA polymerase to long DNA through repeated cycles of folding and melting of the hairpin structure. We discuss the implication of this phenomenon, "hairpin elongation", from the standpoint of potential amplification of simple DNA sequences during the evolution of the genome.     

Purification and characterization of NADP-specific alcohol dehydrogenase and glutamate dehydrogenase from the hyperthermophilic archaeon Thermococcus litoralis.

Thermococcus litoralis is a strictly anaerobic archaeon that grows at temperatures up to 98 degrees C by fermenting peptides. Little is known about the primary metabolic pathways of this organism and, in particular, the role of enzymes that are dependent on thermolabile nicotinamide nucleotides. In this paper we show that the cytoplasmic fraction of cell extracts contained NADP-specific glutamate dehydrogenase (GDH) and NADP-specific alcohol dehydrogenase (ADH) activities, neither of which utilized NAD as a cofactor. The GDH is composed of identical subunits having an M(r) of 45,000 and had an optimal pH and optimal temperature for glutamate oxidation of 8.0 and > 95 degrees C, respectively. Potassium phosphate (60 mM), KCl (300 mM), and NaCl (300 mM) each stimulated the rate of glutamate oxidation activity between two- and threefold. For glutamate oxidation the apparent Km values at 80 degrees C for glutamate and NADP were 0.22 and 0.029 mM, respectively, and for 2-ketoglutarate reduction the apparent Km values for 2-ketoglutarate, NADPH, and NH4+ were 0.16, 0.14, and 0.63 mM, respectively. This enzyme is the first NADP-specific GDH purified form a hyperthermophilic organism. T. litoralis ADH is a tetrameric protein composed of identical subunits having an M(r) of 48,000; the optimal pH and optimal temperature for ethanol oxidation were 8.8 and 80 degrees C, respectively. In contrast to GDH activity, potassium phosphate (60 mM), KCl (0.1 M), and NaCl (0.3 M) inhibited ADH activity, whereas (NH4)2SO4 (0.1 M) had a slight stimulating effect. This enzyme exhibited broad substrate specificity for primary alcohols, but secondary alcohols were not oxidized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elongation of palindromic repetitive DNA by DNA polymerase from hyperthermophilic archaea: a mechanism of DNA elongation and diversification

DNA polymerase from hyperthermophilic bacteria can elongate tandem repetitive oligoDNA with a complete or incomplete palindromic sequence under isothermal conditions by "hairpin elongation". However, the product of the reaction has not yet been sufficiently characterized. Here, I demonstrate that when palindromic repetitive oligoDNA, e.g., (5'AGATATCT3')(6), was added as a "seed" to the DNA synthesis reaction catalyzed by DNA polymerase from the archaea Thermococcus litoralis (Vent polymerase) at 74 degrees C, the product was (5'AGATATCT3')(n). The product itself was palindromic and repetitive, and its motif (unit) sequence was exactly the same as that of the seed oligoDNA. On the other hand, when a pseudopalindrome, which contains a palindrome-breaking nucleotide (underlined), was present in seed oligoDNA, e.g., (5'GATTC3')(6), the product was (5'GATATC3')(n), which had a different motif sequence from that of the seed oligoDNA. When a pseudopalindrome (5'AGATATCA3')(6) was added to the reaction, the products were 5'TATCA . (AGATATCA)(3) . AGATATCT . (TGATATCT)(5) . TGATA3', etc. When 5'AGATATCA . (AGATATCT3')(5) was added, products were 5'TATCT . (AGATATCT)(2).TGATATCT . AGATATCT . AGATATCA . AGATATCT . AGA3', etc., demonstrating the generation of many "mutations" in the product DNA. I conclude that a tandem repetitive sequence is faithfully elongated (amplified) by hyperthermophilic DNA polymerase if it is completely palindromic, but is elongated with many errors if it is incompletely palindromic (pseudopalindromic) or mixed with a pseudopalindrome. The results suggest a protein-catalyzed elongation/diversification mechanism of short repetitive DNAs on the early earth.     

Mechanistic Independence of Avian Myeloblastosis Virus DNA Polymerase and Ribonuclease H

Differential inhibition conditions were established for the DNA polymerase and RNase H activities of avian myeloblastosis virus (AMV) with ether-disrupted AMV and a purified enzyme preparation. The RNase H activity of ether-disrupted AMV with (rA)(n).(dT)(n) and (rA)(n).(dT)(11) as substrates was inhibited 80 to 100% by preincubation with NaF at a final reaction concentration of 27 to 30 mM. Under these conditions, the DNA polymerase activity was inhibited only 0 to 20%. Similar inhibitions were found with exogenous Rous sarcoma virus 35S and 70S RNA.DNA hybrid and phiX174 DNA.RNA hybrid as substrates. Studies were also performed with a purified enzyme preparation, in which the two activities essentially co-purified. The RNase H activity was inhibited >80% by 150 mM KCl with three different hybrid substrates, whereas the DNA polymerase activity was uninhibited. The DNA polymerase was completely inactivated by heat denaturation at 41 C or by omission of the deoxytriphosphates from the reaction mixture; the RNase H remained active. These differential inhibition conditions were used to compare the size of the DNA product synthesized with and without simultaneous RNase H action and to examine the effect of inhibition of the DNA polymerase on the size of the RNase H products. The size of the products of one activity was not affected by inhibition of the other activity. These results suggest that the AMV DNA polymerase and RNase H are not coupled mechanistically.     

DNA-repair reactions by purified HeLa DNA polymerases and exonucleases

PM2 duplex DNA substrates containing small gaps were utilized to study DNA repair reactions of extensively purified HeLa DNase V (a bidirectional double strand DNA exonuclease) and DNA polymerases beta, gamma (mitochondrial and extramitochondrial), and alpha holoenzyme, and delta as a function of ionic strength. At 50 mM NaCl, DNase V carried out extensive exonucleolytic degradation, and beta-polymerase exhibited strand displacement synthesis. However, at 150 mM NaCl, the DNase appeared only to remove damaged nucleotides from DNA termini while beta-polymerase catalyzed only gap-filling synthesis. When present in equimolar amounts, beta-polymerase and DNase V (which can be isolated as a 1:1 complex) catalyzed more degradation than synthesis at 50 mM NaCl; however, at 150 mM NaCl a coupled very limited nick translation reaction ensued. At physiological ionic strength DNA polymerase alpha holoenzyme was not active upon these substrates. In 15 mM KCl it could fill small gaps and carry out limited nick translation with undamaged DNA, but it could not create a ligatable substrate from UV-irradiated DNA incised with T4 UV endonuclease. Mitochondrial DNA polymerase gamma was more active at 150 mM NaCl than at lower ionic strengths. It readily filled small gaps but was only marginally capable of strand-displacement synthesis. The extramitochondrial form of gamma-polymerase, conversely, was less sensitive to ionic strength; it too easily filled small gaps but was not effective in catalyzing strand displacement synthesis. Finally, DNA polymerase delta was able to fill gaps of several to 20 nucleotides in 0.05 M NaCl, but at higher NaCl concentrations there was little activity. DNA polymerases delta did not demonstrate strand displacement synthesis. Therefore, at physiological ionic strength, it appears that either DNA polymerase beta or extramitochondrial DNA polymerase gamma might aid in short patch DNA repair of nuclear (or transfecting) DNAs, whereas mitochondrial gamma-polymerase might fill small gaps in mitochondrial DNA.     

Meiosis in Coprinus: characterization and activities of two forms of DNA polymerase during meiotic stages.

Two forms of DNA polymerase have been studied in the basidiomycete Coprinus. DNA polymerase from basidiocarp tissues at zygotene-pachytene stage has been purified 3,500-fold and defined as DNA polymerase b by virtue of its insensitivity to N-ethylmaleimide and by its low molecular weight (76,000). This enzyme has optimal activity at pH 7.0 to 7.5, at 200 mM KCl, and at 25 degrees C incubation temperature. It can use polycytidylic acid-oligo(dG)12-18 as template primer in addition to homodeoxypolymers. The DNA polymerase a is mainly produced in the exponentially growing mycelium. It is sensitive to N-ethylmaleimide and has a temperature optimum at 35 degrees C. At the premeiotic S phase, activities from both polymerase a and polymerase b are found in cell-free extracts. The b enzyme is the only DNA polymerase produced during meiotic prophase. Its assayable activity exhibits two peaks, one at premeiotic S stage and one at pachytene. It is possible that DNA polymerase b is responsible for pachytene repairs involved in recombination.     

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase--an extremely heat stable enzyme with proofreading activity

We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.     

Characterization of an RNA-directed DNA-polymerase from a cell line derived from a radiation-induced lymphoma in mice.

An RNA-directed DNA polymerase was purified from a cell line derived from a radiation-induced lymphoma in NIH Swiss mice which produced non-infectious type C virus particles. The enzyme was isolated from a high speed particulate fraction which bands at a density of 1.16--1.19 g/ml in a sucrose gradient, and purified by successive chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite. The purified DNA polymerase has a molecular weight of 68 000, a pH optimum of 7.5, a KCl optimum of 50 mM, and a Mn2+ optimum of 0.25 mM. It prefers (dT)15 . (A)n to (dT)15 . (dA)n as the primer template and transcribes the poly(C) strand of (dG)15 .(C)n and (dG)15 . (OMeC)n. It transcribes heteropolymeric regions of avian myeloblastosis virus 70 S RNA, and is inhibited by antiserum to Rauscher murine leukemia virus DNA polymerase. Comparison of the properties of DNA polymerase purified from radiation-induced lymphoma cells with the DNA polymerase purified from non-defective murine type C RNA tumor viruses shows that the mouse lymphoma enzyme is both biochemically and immunologically related to murine leukemia virus DNA polymerases.     

Properties of herpes simplex virus type 1 and type 2 DNA polymerase

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA polymerases were highly purified from infected HeLa BU cells by DEAE cellulose, phosphocellulose and DNA cellulose column chromatography. DNA exonuclease activity but not endonuclease activity was found associated with both types of DNA polymerase. Both DNA polymerase activities could be activated by salt in a similar fashion with the optimal activity in the range of ionic strength between 0.22 and 0.29 alpha. At an ionic strength of 0.14, spermidine and putrescine in the concentration range (0--5 mM) studied could mimic the action of KCI in stimulating DNA polymerase activity. Spermine, in the same concentration range, had a biphasic effect. At an ionic strength of 0.29 all three polyamines were inhibitory. HSV-1 and HSV-2 DNA polymerase are similar in their column chromatographic behavior, sedimentation rate in sucrose gradient centrifugation, and activation energy, but they differ in their heat stability at 45 degrees C with the HSV-2 enzyme more stable than the HSV-1 enzyme. Kinetic behavior of both enzymes is similar, with Km values for deoxyribonucleoside triphosphates in the range of 5 . 10(-7) to 1.8 . 10(-8) M. IdUTP and dUTP served as apparent competitive inhibitors with respect to dTTP, and AraATP acted as an apparent competitive inhibitor with respect to dATP. AraATP could not replace dATP in the DNA polymerization reaction; in contrast, IdUTP could replace TTP. Phosphonoformic acid behaved as an uncompetitive inhibitor with respect to DNA. The ID(50) value estimated was foind to be dependent on the purity of the DNA polymerase used and the ionic strength of the assay condition. Each DNA-polymerase associated DNA exonuclease had the same stability at 45 degrees C as its DNA polymerase. The associated DNAase activity was inhibited by phosphonoformic acid and high ionic strength of the assay condition.     

PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi

DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris-HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 microM of each dNTP. Under these conditions, the error rate was 0.66.10(-6) mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100 degrees C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase (Qbiogene Molecular Biology).     

The interaction in vitro of the intermediate filament protein vimentin with naturally occurring RNAs and DNAs

The binding of the intermediate filament protein vimentin to a variety of naturally occurring RNAs and DNAs was studied. The relative capacities of the various nucleic acids to associate with pure [3H]vimentin were determined in competition experiments with 28 S rRNA from Ehrlich ascites tumor cells. The reaction products were analyzed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. Under these ionic conditions, vimentin reacted preferentially with single-stranded nucleic acids, particularly with those of high (G + C) content. The vimentin binding potentials of single-stranded RNAs and DNAs were largely comparable. However, when the concentrations of mono- and divalent cations were raised to physiological and higher values, only single-stranded DNA retained its vimentin binding capacity. With increasing KCl concentrations at 0 to 1 mM Mg2+, increasing amounts of vimentin were detected in complexes which sedimented considerably faster than the bulk of the DNA, suggesting cooperative binding of vimentin. The salt optimum of this cooperativity was at 200 mM KCl. Thus, the capability of vimentin to discriminate between single-stranded RNA and DNA under physiological ionic conditions points to specificity of the interaction of vimentin with nucleic acids.     

Photoelectric signals generated by bovine rod outer segment disk membranes attached to a lecithin bilayer.

Purified bovine rod outer segment disk membranes were attached to a lecithin bilayer membrane. After photoexcitation with a 500-nm flash delivered by a dye laser, a negative photovoltage was observed on the bilayer under normal ionic strengths (100 mM KCl), which had a rise phase of 1-3 ms at 20 degrees C. The photoresponse was obviously due to bleaching of rhodopsin as it decreased for successive flashes of light. It originated most probably during the metarhodopsin-I metarhodopsin-II (meta-I-II) transition of rhodopsin because it was pH dependent at 2 degrees C but not at 20 degrees C. At 10 mM KCl, i.e., under hypotonic conditions, a positive photovoltage with slower kinetics than at high salt was observed. As the disk membranes were merely attached to the bilayer membrane, the photovoltage was apparently due to a light-induced transmembrane potential change in the disk membranes. Possible electrogenic mechanisms underlying the photosignal will be discussed.     

Bovine cardiac myosin subfragment 1. Transient kinetics of ATP hydrolysis

The kinetics of binding and hydrolysis of ATP by bovine cardiac myosin subfragment 1 has been reinvestigated. More than 90% of the total fluorescence amplitude associated with ATP hydrolysis occurs with an apparent second-order rate constant of 8.1 X 10(5) M-1 S-1 and a limiting rate constant of approximately 140 S-1 (100 mM KCl, 50 mM 1,3-bis-[tris(hydroxymethyl)methylamino]-propane, 10 mM MgCl2, pH 7.0, 20 degrees C); the remaining 10% occurs more slowly (approximately 1 S-1). The observed rate constants are independent of subfragment 1 concentration under pseudo first-order conditions for ATP with respect to protein. The fraction of protein which hydrolyzes ATP rapidly is not a function of the nucleotide or protein concentration and appears to be constant irrespective of ionic strength or temperature within the range studied (50-100 mM KCl, pH 7.0, 15-20 degrees C). These data are compared to that obtained previously using subfragment 1 prepared by a different method which showed ATP-dependent aggregation of two protein species.