Molecular diversity and catalytic activity of Thermus DNA polymerases

Thermus aquaticus DNA polymerase (Taq polymerase) made the polymerase chain reaction feasible and led to a paradigm shift in genomic analysis. Other Thermus polymerases were reported to have comparable performance in PCR and there was an analysis of their properties in the 1990s. We re-evaluated our earlier phylogeny of Thermus species on the basis of 16S rDNA sequences and concluded that the genus could be divided into eight clades. We examined 22 representative isolates and isolated their DNA polymerase I genes. The eight most diverse polymerase genes were selected to represent the eight clades and cloned into an expression vector coding for a His-tag. Six of the eight polymerases were expressed so that there was sufficient protein for purification. The proteins were purified to homogeneity and examination of the biochemical characteristics showed that although they were competent to perform PCR, none was as thermostable as commercially available Taq polymerase; all had similar error-frequencies to Taq polymerase and all showed the expected 5′–3′ exonuclease activity. We conclude that the initial selection of T. aquaticus for DNA polymerase purification was a far-reaching and fortuitous choice but simple mutagenesis procedures on other Thermus-derived polymerases should provide comparable thermostability for the PCR reaction.     

RACE using only a gene-specific primer

This article describes a simple method for accurate rapid amplification of complementary deoxyribonucleic acid (cDNA) ends (RACE), the distinctive feature being that only a gene-specific primer is used, without an anchor or adapter primer. Under these conditions, Thermus aquaticus (Taq) polymerase synthesizes cDNA ends exactly, so that amplified products obtain a characteristic structure: a terminal inverted repeat composed of a gene-specific primer and occasionally several nucleotides from its 3′ flanking sequence. These structures suggest a hypothetical mechanism of cDNA end synthesis in which Taq DNA polymerase synthesizes a sequence complementary to the gene-specific primer at the 3′ end of the daughter strand by switching the template to the 5′ terminal region through circularization of the DNA. As a result, the targeted cDNA will be efficiently amplified with only a single gene-specific primer. This technique, which provides highly specific amplification of the 5′ and 3′ ends of a cDNA, is especially useful for isolation of cDNA when the corresponding messenger ribonucleic acid is scarce.     

Synthesis and biological properties of pyrimidine 4′-fluoronucleosides and 4′-fluorouridine 5′-<Emphasis Type="Italic">O</Emphasis>-triphosphate

4′-Fluoro-2′,3′-O-isopropylidenecytidine was synthesized by the treatment of 5′-O-acetyl-4′-fluoro-2′,3′-O-isopropylideneuridine with triazole and 4-chlorophenyl dichlorophosphate followed by ammonolysis. The interaction of 4′-fluoro-2′,3′-O-isopropylidenecytidine with hydroxylamine resulted in 4′-fluoro-2′,3′-O-isopropylidene-5′-O-acetyl-N ⁴-hydroxycytidine. The removal of the 2′,3′-O-isopropylidene groups led to acetyl derivatives of 4′-fluorouridine, 4′-fluorocytidine, and 4′-fluoro-N ⁴-hydroxycytidine. 4′-Fluorouridine 5′-O-triphosphate was obtained in three steps starting from 4′-fluoro-2′,3′-O-isopropylideneuridine. 4′-Fluorouridine 5′-O-triphosphate was shown to be an effective inhibitor of HCV RNA-dependent RNA polymerase and a substrate for the NTPase reaction catalyzed by the HCV NS3 protein, the hydrolysis rate being similar to that of ATP. It could also activate a helicase reaction with an efficacy of only threefold lower than that for ATP.     

A critical evaluation of metal-promoted Klenow 3′-5′ exonuclease activity: calorimetric and kinetic analyses support a one-metal-ion mechanism

 Metal-mediated hydrolysis of phosphate esters is a common catalytic pathway in nucleic acid biochemistry. Two distinct models are principally invoked in mechanistic discussions of these reactions for magnesium-dependent nuclease activation: namely, the one-versus two-metal-ion pathways. The 3′-5′ exonuclease domain of the Klenow fragment of Escherichia coli DNA polymerase I is a paradigm for the two-metal-ion mechanism; however, this reaction model is principally based on structural and kinetics experiments employing high concentrations of transition metal analogues and high concentrations of background ammonium sulfate during doping experiments. This prompted us to re-evaluate the metal cofactor stoichiometry of the 3′-5′ exonuclease mechanism for the Klenow fragment by solution kinetics and isothermal titration calorimetry using the natural Mg²⁺ cofactor and salt conditions. Both solution calorimetric and kinetics experiments strongly indicate binding of only one metal ion to the exonuclease active site. Comparative studies with Mn²⁺ also indicate a requirement for one metal ion to effect 3′-5′ exonuclease activity. Received, accepted: 16 March 1998     

Purification and characterization of UDP-glucose: anthocyanin 3′,5′-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin 3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable k _cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K _m value for 1 (3.89 × 10⁻⁵ M), which is lower than that for 2 (1.38 × 10⁻⁴ M), renders the k _cat/K _m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K _m value for UDP-glucose (6.18 × 10⁻³ M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10⁻³ M) with saturated 1, the k _cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center. K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work.     

<Emphasis Type="Italic">SpnH</Emphasis> from <Emphasis Type="Italic">Saccharopolyspora spinosa</Emphasis> encodes a rhamnosyl 4′-<Emphasis Type="Italic">O</Emphasis>-methyltransferase for biosynthesis of the insecticidal macrolide,spinosyn A

Deoxysugar, 2′, 3′, 4′-tri-O-methylrhamnose is an essential structural component of spinosyn A and D, which are the active ingredients of the commercial insect control agent, Spinosad. The spnH gene, which was previously assigned as a rhamnose O-methyltransferase based on gene sequence homology, was cloned from the wild-type Saccharopolyspora spinosa and from a spinosyn K-producing mutant that was defective in the 4′-O-methylation of 2′, 3′-tri-O-methylrhamnose. DNA sequencing confirmed a mutation resulting in an amino acid substitution of G-165 to A-165 in the rhamnosyl 4′-O-methyltransferase of the mutant strain, and the subsequent sequence analysis showed that the mutation occurred in a highly conserved region of the translated amino acid sequence. Both spnH and the gene defective in 4′-O-methylation activity (spnH165A) were expressed heterologously in E. coli and were then purified to homogeneity using a His-tag affinity column. Substrate bioconversion studies showed that the enzyme encoded by spnH, but not spnH165A, could utilize spinosyn K as a substrate. When the wild-type spnH gene was transformed into the spinosyn K-producing mutant, spinosyn A production was restored. These results establish that the enzyme encoded by the spnH gene in wild-type S. spinosa is a rhamnosyl 4′-O-methyltransferase that is responsible for the final rhamnosyl methylation step in the biosynthesis of spinosyn A.     

News & Notes: Primary Structure of the DNA Polymerase I Gene of an α-Proteobacterium, Rhizobium leguminosarum, and Comparison with Other Family A DNA Polymerases

The structural gene for DNA polymerase I of Rhizobium leguminosarum was determined. The rhizobium DNA polymerase I consists of 1016 amino acid residues with a calculated molecular weight of 111,491 Dalton. The amino acid sequence comparison with E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, and Rickettsia prowazekii DNA polymerase I showed that, although 5′-nuclease and DNA polymerase domains are highly conserved, 3′ to 5′ exonuclease domains are much less conserved. While both R. leguminosarum and R. prowazekii belong to the alpha subdivision of the Proteobacteria on the basis of 16S ribosomal RNA phylogeny, the primary structure of the DNA polymerase I is quite different; the rhizobium DNA polymerase I has 3′ to 5′ proofreading exonuclease, but the rickettsia DNA polymerase I does not. Received: 15 December 1998 / Accepted: 2 February 1999     

Cloning,expression, and PCR application of DNA polymerase from the hyperthermophilic archaeon, <Emphasis Type="Italic">Thermococcus celer</Emphasis>

The family B DNA polymerase gene was amplified from Thermococcus celer genomic DNA by using the degenerate primers and DNA walking PCR. The Tce DNA polymerase gene was cloned and sequenced. The gene contains an ORF of 2,325 bp encoding 774 amino acid residues with a calculated molecular weight of 89,788.9 kDa. The Tce DNA polymerase was purified by heat treatment and heparin column chromatography. The optimal conditions for PCR were determined. Long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tce DNA polymerases (Tce plus DNA polymerase). Tce plus DNA polymerase surpassed the PCR performance of Tce, Taq and Pfu DNA polymerases in terms of yield and efficiency.     

Isolation and potential cancer chemopreventive activities of phenolic compounds of beer

Beer contains a variety of phenolic compounds. During the brewing process, some of these compounds are removed by polyvinylpolypyrrolidone (PVPP) to prevent haze formation. We have analyzed the phytochemical composition of a PVPP residue as well as of unstabilized beer and isolated a total of 51 compounds. Eight structures were identified as novel, i.e., 2-(4′-hydroxyphenyl)-3,5-dihydroxybenzoic acid (6), 2′-(4″-hydroxyphenyl)isoferulic acid ester (12), 1,2,5,7-tetrahydroxyanthraquinone (23) and 4,7-dihydroxy-5-(2′,4′,6′-trihydroxyphenyl)-indan-1,2-dione (24) from the PVPP residue, and catechin-7-O-β-(6″-O-nicotinoyl)-β-D-glucopyranoside (41), ent-epigallo-catechin-(4αto8, 2αtoOto7)catechin (44), ent-epigallocatechin (4αto6, 2αtoOto7)catechin (45) and 2,3-cis-3,4-trans-2-[2,3-trans-3,3′,4′,5,7-pentahydroxyflavan-8-yl]-4-(3,4-dihydroxyphenyl)3,5,7-trihydroxybenzopyran (46) from the unstabilized beer. Most of the compounds were tested for potential cancer chemopreventive activities in in vitro test systems detecting a modulation of carcinogen metabolism (inhibition of phase 1 cytochrome P450 1A (Cyp1A) activity, induction of NAD(P)H:quinone oxidoreductase (QR) activity) and anti-inflammatory mechanisms (inhibition of lipopolysaccharide (LPS)-mediated induction of inducible nitric oxide synthase (iNOS), inhibition of cyclooxygenase 1 (Cox-1) activity). 1,2,5,7-Tetrahydroxyanthraquinone (23) and xanthohumol (25), a prenylated chalcone derived from hop, were identified as the most potent compounds and were additionally tested for inhibition of chemically-induced preneoplastic lesions in an ex vivo mouse mammary gland organ culture model (MMOC). Importantly, both agents inhibited lesion formation with halfmaximal inhibitory concentrations (IC₅₀) of 0.1 and 0.02 μM, respectively. Our results demonstrate that beer is an interesting source of potential cancer chemopreventive agents and should be further investigated with this respect. This revised version was published online in June 2006 with corrections to the Cover Date.     

Targeting topoisomerase II with the chiral DNA-intercalating ruthenium(II) polypyridyl complexes

Many antitumor drugs act as topoisomerase inhibitors, and the inhibitions are usually related to DNA binding. Here we designed and synthesized DNA-intercalating Ru(II) polypyridyl complexes Δ--[Ru(bpy)₂(uip)]²⁺ and Λ-[Ru(bpy)₂(uip)]²⁺ (bpy is 2,2′-bipyridyl, uip is 2-(5-uracil)-1H-imidazo[4,5-f][1,10]phenanthroline). The DNA binding, photocleavage, topoisomerase inhibition, and cytotoxicity of the complexes were studied. As we expected, the synthesized Ru(II) complexes can intercalate into DNA base pairs and cleave the pBR322 DNA with high activity upon irradiation. The mechanism studies reveal that singlet oxygen (¹O₂) and superoxide anion radical (O₂^•−) may play an important role in the photocleavage. The inhibition of topoisomerases I and II by the Ru(II) complexes has been studied. The results suggest that both complexes are efficient inhibitors towards topoisomerase II by interference with the DNA religation and direct topoisomerase II binding. Both complexes show antitumor activity towards HELA, hepG2, BEL-7402, and CNE-1 tumor cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biochemical properties of a plastidial DNA polymerase of rice

Plastids are organelles unique to plant cells and are responsible for photosynthesis and other metabolic functions. Despite their important cellular roles, relatively little is known about the mechanism of plastidial DNA replication and repair. Recently, we identified a novel DNA polymerase in Oryza Sativa L. (OsPOLP1, formerly termed OsPolI-like) that is homologous to prokaryotic DNA polymerase Is (PolIs), and suggested that this polymerase might be involved in plastidial DNA replication and repair. Here, we propose to rename the plant PolI homologs as DNA polymerase π (POLP), and investigate the biochemical properties of full-length OsPOLP1. The purified OsPOLP1 elongated both DNA and RNA primer hybridized to a DNA template, and possessed a 3′ exonuclease activity. Moreover, OsPOLP1 displayed high processivity and fidelity, indicating that this polymerase has the biochemical characteristics appropriate for DNA replication. We found that POLPs have two extra sequences in the polymerase domain that are absent in prokaryotic PolIs. Deletion of either insert from OsPOLP1 caused a decrease in DNA synthetic activity, processivity, and DNA binding activity. In addition, OsPOLP1 efficiently catalyzed strand displacement on nicked DNA with a 5′-deoxyribose phosphate, suggesting that this enzyme might be involved in a repair pathway similar to long-patch base excision repair. These results provide insights into the possible role of POLPs in plastidial DNA replication and repair. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20–50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq DNA pol/TBD is thermostable, PCR competent and able to copy repetitive deoxynucleotide sequences six to seven times more faithfully than Taq DNA polymerase and makes 2–3-fold fewer AT→GC transition mutations.     

Domain organization and biochemical features of Sulfolobus solfataricus DNA polymerase

DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization of its associated catalytic functions (DNA polymerase and 3′-5′ exonuclease). Indeed, the synthetic function was ascribed to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity. In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better investigate the role of these two protein segments in DNA substrate interaction. Received: January 22, 1998 / Accepted: February 16, 1998     

The preparation of a photoreactive analogue of 2′,3′-dideoxyuridine 5′-triphosphate and its use for photoaffinity modification of human replication protein A

A new reagent for photoaffinity modification of biopolymers, 5-[E-N-(2-nitro-5-azidobenzoyl)-3-amino-1-propen-1-yl]-2′,3′-dideoxyuridine 5′-triphosphate (NAB-ddUTP), was synthesized. Like a similar derivative of 2′-deoxyuridine 5′-triphosphate (NAB-dUTP), it was shown to be able to effectively substitute for dTTP in the synthesis of DNA catalyzed by eukaryotic DNA polymerase β and to terminate DNA synthesis. A 5′-³²P-labeled primer with a photoreactive group at the 3′-terminus was derived from NAB-ddUTP and used for photoaffinity labeling of the human replication protein A (RPA). The covalent attachment of RPA p32 and p70 subunits to the labeled primers was demonstrated. NAB-ddUTP is a promising tool for studying the interaction of proteins of the replicative complex with NA in cellular extracts and living cells during the termination of DNA synthesis.     

A probe for detection of G-rich target strands through fluorescence quenching

A modified fluorescent probe U^FAA AAT CTC CGC CGC was synthesized using the nucleoside analogue 3′-O-(N,N′-diisopropylamino-2-cyanoethoxyphosphinyl)-5′-O-(4,4′-dimethoxytrityl)-2′-O-(dansyl-1-sulfonamidohexylaminocarbonyl)uridine for hybridization studies with perfectly matched (U/A) complementary DNA and with a DNA strand having similar G-rich telomeric units at their 3′-ends. Data on the thermal stability and decrease in fluorescence intensity due to the presence of dG units clearly demonstrated the potential application of this approach in DNA diagnostics in homogeneous hybridization assays. The text was submitted by the authors in English.     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

TspMI, a thermostable isoschizomer of XmaI (5′C/CCGGG3′): characterization and single molecule imaging with DNA

TspMI, a thermostable isoschizomer of XmaI from a Thermus sp., has been characterized. The enzyme was purified to homogeneity using Cibacron-Blue 3GA agarose, Heparin agarose, SP sephadex C50, and Mono-Q fast protein liquid chromatography and was found to be a homodimer of 40 kDa. Restriction mapping and run-off sequencing of TspMI-cleaved DNA ends depicted that it cleaved at 5′C/CCGGG3′ to generate a four-base, 5′-CCGG overhang. The enzyme was sensitive to methylation of second and third cytosines in its recognition sequence. TspMI worked optimally at 60°C with 6 mM Mg²⁺, no Na⁺/K⁺, and showed no star activity in the presence of 25% glycerol. The enzyme could efficiently digest the DNA labeled with a higher concentration of YOYO-I (one dye molecule to one nucleotide), making it a useful candidate for real-time imaging experiments. Single molecule interaction between TspMI and λ DNA was studied using total internal reflection fluorescence microscopy. The enzyme survived 30 polymerase chain reaction (PCR) cycles in the presence of 10% glycerol and 0.5 M trehalose without any activity loss and, hence, is suitable for incorporation in restriction-endonuclease-mediated selective-PCR for various applications.Electronic Supplementary Material Supplementary material for this article is available at     

Characterization of new glycolipid biosurfactants,tri-acylated mannosylerythritol lipids,produced by <Emphasis Type="Italic">Pseudozyma</Emphasis> yeasts

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by Pseudozyma yeasts. They show not only the excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from soybean oil by P. antarctica and P. rugulosa, some new extracellular glycolipids (more hydrophobic than the previously reported di-acylated MELs) were found in the culture medium. The most hydrophobic one was identified as 1-O-alka(e)noyl-4-O-[(4′,6′-di-O-acetyl-2′,3′-di-O-alka(e)noyl)-β-d-mannopyranosyl]-d-erythritol, namely tri-acylated MEL. Others were tri-acylated MELs bearing only one acetyl group. The tri-acylated MEL could be prepared by the lipase-catalyzed esterification of a di-acylated MEL with oleic acid implying that the new glycolipids are synthesized from di-acylated MELs in the culture medium containing the residual fatty acids.     

New morphotypes of condensed DNA microparticles formed in PCR with <Emphasis Type="Italic">KlenTaq</Emphasis> and <Emphasis Type="Italic">Taq</Emphasis> polymerases and plasmid DNA as a template

The specimens of DNA microparticles formed during PCR amplification of IS-elements ISAfe1 and IST2 by KlenTaq or Taq polymerases and plasmid DNA as a template under varying conditions were investigated by electron microscopy. Microparticle yield and morphology were found to depend on the level of synthesis of single-stranded DNA fragments during PCR. The conditions were studied for formation of discs (ellipsoids) several micrometers in diameter and several dozens of nanometers thick, as well as of microparticles of other morphologies, in the course of PCR with Taq polymerase. The structure of the microparticles produced during an asymmetric PCR, i.e., under conditions of low concentration of one of the two primers, was investigated. Morphology of the DNA micro- and nanoparticles was found to depend mainly on the DNA polymerase used in asymmetric PCR. In particular, in the presence of the KlenTaq polymerase, discs or ellipsoids a few dozen nanometers thick were formed, while in the presence of the Taq polymerase, micro- and nanospheres, heterogeneous in size with rugged surfaces, were produced. The effect of Mn²⁺ cations on DNA microparticle morphology was studied. In the presence of Mn²⁺, microparticle morphology changed dramatically; in PCR mixtures containing KlenTaq polymerase supplemented with Mn²⁺, DNA microspheres with fringed surfaces were formed; in the presence of Taq polymerase, microparticles in the form of short, rounded rods were produced. In light of these data, the molecular mechanism of micro- and nanoparticle formation in the course of PCR is discussed.