New DNA polymerase from the hyperthermophilic marine archaeon <Emphasis Type="Italic">Thermococcus thioreducens</Emphasis>

The family B DNA polymerase gene of Thermococcus thioreducens, an archaeon recently isolated from the Rainbow hydrothermal vent field, was cloned and its protein product expressed, purified and characterized. The gene was found to encode a 1,311 amino acid chain including an intein sequence of 537 residues. Phylogenetic analysis revealed a predominantly vertical type of inheritance of the intein in the Thermococcales order. Primary sequence analysis of the mature protein (TthiPolB) showed significant sequence conservation among DNA polymerases in this family. The structural fold of TthiPolB was predicted against the known crystallographic structure of a family B DNA polymerase from Thermococcus gorgonarius, allowing regional domain assignments within the TthiPolB sequence. The recombinant TthiPolB was overexpressed in Escherichia coli and purified for biochemical characterization. Compared with other DNA polymerases from the Thermococcales order, TthiPolB was found to have moderate thermal stability and fidelity, and a high extension rate, consistent with an extremely low K _m corresponding to the dNTP substrate. TthiPolB performed remarkably well in a wide range of PCR conditions, being faster, more stable and more accurate than many commonly used enzymes.     

Detection of Rhopalosiphum insertum (apple-grass aphid) predation by the predatory mite Anystis baccarum using molecular gut analysis

Abstract 1 A simple, yet sensitive polymerase chain reaction based technique was developed for the detection of the apple‐grass aphid Rhopalosiphum insertum in the gut of Anystis baccarum, a predatory mite. 2 A range of conserved polymerase chain reaction primers for insect mitochondrial and ribosomal DNA were tested in order to amplify R. insertum DNA. The mitochondrial DNA primers LrRNAR2 + N1F1, amplified a region between the ND1 and large subunit RNA genes. 3 DNA sequencing of the R. insertum ND1‐LRNA polymerase chain reaction product allowed aphid‐specific polymerase chain reaction primers to be designed. These amplified a 283‐bp product from individual aphids. No polymerase chain reaction product was amplified from individual A. baccarum. 4 Using the aphid‐specific primers against A. baccarum fed on R. insertum, the diagnostic 283‐bp product was amplified. 5 Two restriction enzymes (RsaI and AluI) produced patterns that allowed unambiguous identification of R. insertum DNA from that of Macrosiphum euphorbiae and Myzus persicae.     

Some biochemical properties of an acyclic oligonucleotide analogue. A plausible ancestor of the DNA?

As acyclic oligonucleotides have been suggested as a primitive model of DNA or RNA in prebiotic times, we compared some biochemical properties of these analogues to that of natural ones. Firstly, an acyclic analogue of deoxyribonucleoside triphosphates was tested as a potential substrate of enzymes intervening in nucleic acids synthesis. GlyTTP, a dTTP analogue with a missing 2-methylene group is notaccepted as a substrate by either DNA polymerase or deoxynucleotidyl terminal transferase (TdT). Secondly, themodified dodecathymidylate (GlyT)₁₂, the racemic acyclic sugar analogue of (dT)₁₂, proved to be anefficient primer for DNA polymerase and TdT, though the associative properties of (GlyT)₁₂ are very weak as shown by UV spectroscopy in phosphate buffer without magnesium chloride. But (GlyT)₁₂ has the advantage to be 500-times more stable against hydrolysis by snake venom phosphodiesterase than the corresponding oligothymidylate.     

Prostaglandin D2 lowers nuclear DNA polymerase activity in cultured mastocytoma cells

Prostaglandin D₂ strongly inhibited growth of cultured mastocytoma P-815, 2-E-6 cells, which were established and cloned from mouse mast tumor cells. The inhibition was dose-dependent (IC₅₀ = 2.09 × 10⁻⁵ M). Prostaglandin D₂ also inhibited the DNA synthesizing activity of the cells dose-dependently. We next measured the activities of endogenous DNA polymerases extracted from untreated and prostaglandin D₂-treated cells. Prostaglandin D₂ pretreatment reduced DNA polymerase α activity by 52%. The sedimentation coefficients of the enzymes from untreated and prostaglandin D₂-treated cells were the same suggesting there was no gross change in the size of the enzyme. Prostaglandin D₂ pretreatment of the cells reduced endogenous DNA polymerase β activity to 68% of the control value; the sedimentation coefficients of the enzymes from treated and untreated cells were both 3.5 S. Interestingly, prostaglandin D₂ had no direct inhibitory effect on the activity of either DNA polymerase α or β. Our results indicate that the activities of DNA polymerase α and β are lower in prostaglandin D₂-treated mastocytoma cells. This finding account for the lower level of DNA synthesis in these cells.     

Chinese hamster ovary cell mutants resistant to DNA polymerase inhibitors

Summary Isolation and characterization of Chinese hamster ovary cell mutants resistant to different DNA polymerase ase inhibitors (aphidicolin, ara-A and ara-C) have been described. A particular mutant (JK3-1-2A) characterized in detail was found to grow and synthesize DNA in medium containing an amount of aphidicolin tenfold greater than that which completely inhibited the growth and the DNA synthesis of the wild-type cells. An almost twofold increase in the specific activity of the DNA polymerase was seen in this mutant. The mutant DNA polymerase showed altered aphidicolin inhibition kinetics of dCMP incorporation; the apparent K _m for dCTP and the apparent K _i for aphidicolin were increased in the mutant. These alterations in the kinetic parameters were, however, abolished upon further purification of the enzyme. Ara-CTP was found to act as a competitive inhibitor of the dCMP incorporation by both the wild type and mutant enzymes. In contrast, the effect of aphidicolin on dCMP incorporation was either competitive (wild-type enzymes) or noncompetitive (mutant enzyme). The data presented showed that the sites of action for aphidicolin and ara-CTP were distinct; likewise the dCTP binding site appeared to be separate from other dNTP(s) binding sites. The drug resistance of the mutant was inherited as a dominant trait.Abbreviations ara-A 9--d-arabinofuranosyl adenine - ara-C 1--d-arabinofuranosyl cytosine - aph aphidicolin     

Characterization of DNA polymerase from the hyperthermophilic archaeon Thermococcus marinus and its application to PCR

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap™ Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris–HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH₄)₂SO_4, 2 mM MgCl_2, 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).     

Sensitivity of Bacteriophage RB69 DNA Polymerase to n2-(p-n-Butylphenyl)-2′-Deoxyguanosinenucleotides

Abstract

The response of bacteriophage RB69 DNA polymerase to N²-(p-n-butylphenyl)-2′-deoxyguanosine 5′-triphosphate (BuPdGTP), related nucleotides and non-nucleoside inhibitors was measured and compared to values obtained for the closely related DNA polymerase from bacteriophage T4. Both enzymes showed similar responses to inhibitors in terms of K_i values and the ability to utilize BuPdGTP as a substrate. These results provide the relevance of using the recent crystal structure of RB69 DNA polymerase for analysis of BuPdGTP/B family DNA polymerase interactions.     

Calcium Ion-Dependent Increase in Thermostability of Dextran Glucosidase from Streptococcus mutans

Dextran glucosidase from Streptococcus mutans (SmDG), which belongs to glycoside hydrolase family 13 (GH13), hydrolyzes the non-reducing terminal glucosidic linkage of isomaltooligosaccharides and dextran. Thermal deactivation of SmDG did not follow the single exponential decay but rather the two-step irreversible deactivation model, which involves an active intermediate having 39% specific activity. The presence of a low concentration of CaCl₂ increased the thermostability of SmDG, mainly due to a marked reduction in the rate constant of deactivation of the intermediate. The addition of MgCl₂ also enhanced thermostability, while KCl and NaCl were not effective. Therefore, divalent cations, particularly Ca²⁺, were considered to stabilize SmDG. On the other hand, CaCl₂ had no significant effect on catalytic reaction. The enhanced stability by Ca²⁺ was probably related to calcium binding in the β→α loop 1 of the (β?α)₈ barrel of SmDG. Because similar structures and sequences are widespread in GH13, these GH13 enzymes might have been stabilized by calcium ions.     

Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum

Mitochondria of chloroquine-resistant Plasmodium falciparum (K1 strain) were isolated from mature trophozoites by differential centrifugation. The mitochondrial marker enzyme cytochrome c reductase was employed to monitor the steps of mitochondria isolation. Partial purification of DNA polymerase from P. falciparum mitochondria was performed using fast protein liquid chromatography (FPLC). DNA polymerase of P. falciparum mitochondria was characterized as a γ-like DNA polymerase based on its sensitivity to the inhibitors aphidicolin, N-ethylmaleimide and 9-β- -arabinofuranosyladenine-5′-triphosphate. In contrast, the enzyme was found to be strongly resistant to 2′,3′-dideoxythymidine-5′-triphosphate (IC₅₀>400 μM) and differed in this aspect from the human homologue, possibly indicating structural differences between human and P. falciparum DNA polymerase γ. In addition, the DNA polymerase of parasite mitochondria was shown to be resistant (IC₅₀>1 mM) to the nucleotide analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]adenine diphosphate (HPMPApp).     

Substituted 4-hydroxyproline di- and tri-peptides as cytotoxic agents

Summary 4-Hydroxyproline di- and tri-peptides and N-cbz-hydroxypropylglycinamides were observed to be potent cytotoxic agents against the growth of suspended single cells, L-1210, Tmolt₃, and HeLa-S³. The agents were not as potent against the growth of cultured solid tumor cells. Selected derivatives were investigated for their mode of action in Tmolt₃ leukemia cells. The compounds selectively inhibited DNA synthesis at 50 and 100smM. The target site of action of the agents appeared to be the purinede novo pathway with marked inhibition of the activities of the two regulatory enzymes of the pathway, i.e. PRPP amido-transferase and IMP dehydrogenase. d[NTP] pools were reduced by the agents consistent with their overall reduction of DNA synthesis. Other marginally inhibited targets of the agents were r-RNA polymerase and TMP-kinase activities. The DNA molecule itself did not appear to be a target of these agents.     

Differential inactivation of DNA polymerases α and β by aldehyde compounds

The effects of cyclohexanecarboxaldehyde, benzaldehyde and protocatechualdehyde on the activities of DNA polymerases α, β and E. coli DNA polymerase I were investigated. On direct addition of the aldehydes to the DNA polymerase assay mixture containing activated DNA or poly(dA) (dT)_12–18 as a template, DNA polymerase α was most strongly inhibited by the aldehyde compounds, while DNA polymerases β and I were resistant to such aldehyde inhibition. On preincubation of the enzymes with aldehyde, both DNA polymerases α and β were inactivated; however, DNA polymerase β was protected from the inactivation when activated DNA was added to the preincubation mixture. The inhibition of DNA polymerase α by aldehyde was noncompetitive with regard to the substrate dNTP and competitive with regard to the template DNA. The extent of inhibition of DNA polymerase α by aldehyde was partly reduced by the addition of cysteine to the reaction mixture.     

Functional characterization of Class II 5-enopyruvylshikimate-3-phosphate synthase from <Emphasis Type="Italic">Halothermothrix orenii</Emphasis> H168 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Although a large number of AroA enzymes (5-enopyruvylshikimate-3-phosphate synthase [EPSPS]) have been identified, cloned and tested for glyphosate resistance, only AroA variants derived from Agrobacterium tumefaciens strain CP4 have been successfully used commercially. We have now used a polymerase chain reaction (PCR)-based two-step DNA synthesis (PTDS) method to synthesize an aroA gene (aroA _{H. orenii} ) from Halothermothrix orenii H168 encoding a new EPSPS similar to AroA _{A. tumefaciens CP4.} AroA _{H. orenii} was then expressed in Escherichia coli and key kinetic values of the purified enzyme were determined. Kinetic analysis of AroA _{H. orenii} indicated that the full-length enzyme exhibited increased tolerance to glyphosate compared with E. coli AroA _{E. coli} while retaining a high affinity for the substrate phosphoenolpyruvate. Transgenic Arabidopsis plants containing aroA _{H. orenii} were resistant to 15 mM glyphosate. Site-directed mutagenesis showed that residues Thr355Ser affected the affinity of AroA _{H. orenii} for glyphosate, providing further evidence that specific amino acid residues are responsible for differences in enzymatic behavior among different AroA enzymes.     

Chloroplast and mitochondrial DNA polymerases from cultured soybean cells

DNA polymerases were purified from chloroplasts and mitochondria of cultured Glycine max cells. The chloroplast enzyme exists in two forms which are indistinguishable from each other biochemically. All three organellar enzymes have an estimated molecular weight of 85,000 to 90,000 and prefer poly(rA)dT_12-18 over activated DNA as a template in vitro. Maximum activity of the chloroplast and mitochondrial DNA polymerases requires KCl and a reducing agent, and the enzymes are completely resistant to inhibitors of DNA polymerase α. Taken together, these properties classify the soybean organellar enzymes as DNA polymerases γ. A unique feature that distinguishes the plant enzymes from their animal counterparts is their resistance to dideoxyribonucleotides.     

Metabolic pathway of 6-aminohexanoate in the nylon oligomer-degrading bacterium Arthrobacter sp. KI72: identification of the enzymes responsible for the conversion of 6-aminohexanoate to adipate

Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD ₁ and nylE ₁ , responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies. We confirmed that 6-aminohexanoate aminotransferase (NylD₁) catalyzes the reaction of 6-aminohexanoate to adipate semialdehyde using α-ketoglutarate, pyruvate, and glyoxylate as amino acceptors, generating glutamate, alanine, and glycine, respectively. The reaction requires pyridoxal phosphate (PLP) as a cofactor. For further metabolism, adipate semialdehyde dehydrogenase (NylE₁) catalyzes the oxidative reaction of adipate semialdehyde to adipate using NADP⁺ as a cofactor. Phylogenic analysis revealed that NylD₁ should be placed in a branch of the PLP-dependent aminotransferase sub III, while NylE₁ should be in a branch of the aldehyde dehydrogenase superfamily. In addition, we established a NylD₁/NylE₁ coupled system to quantify the aminotransferase activity and to enable the conversion of 6-aminohexaoate to adipate via adipate semialdehyde with a yield of > 90%. In the present study, we demonstrate that 6-aminohexanoate produced from polymeric nylon-6 and nylon oligomers (i.e., a mixture of 6-aminohexaoate oligomers) by nylon hydrolase (NylC) and 6-aminohexanoate dimer hydrolase (NylB) reactions are sequentially converted to adipate by metabolic engineering technology.

     

A caveat for the use of log P values for the assessment of the biocompatibility of organic solvents

The degree of enzyme deactivation for lipases from Candida rugosa and Pseudomonas sp., hydroxynitrile lyase and mandelate racemase upon exposure to organic solvents can be correlated to their respective partition coefficients (log P values). However, three unexpected results were obtained: (1) the deactivation exerted by protic solvents, e.g., methanol, is severely underestimated; (2) little deactivation by an organic solvent cannot neccessarily be correlated to catalytic activity in this medium, and (3) in contrast to other enzymes, hydroxynitrile lyase is exceptionally stable towards deactivation by DMF.     

Screening for novel lipolytic enzymes from uncultured soil microorganisms

The construction and screening of metagenomic libraries constitute a valuable resource for obtaining novel biocatalysts. In this work, we present the construction of a metagenomic library in Escherichia coli using fosmid and microbial DNA directly isolated from forest topsoil and screened for lipolytic enzymes. The library consisted of 33,700 clones with an average DNA insert size of 35 kb. Eight unique lipolytic active clones were obtained from the metagenomic library on the basis of tributyrin hydrolysis. Subsequently, secondary libraries in a high-copy-number plasmid were generated to select lipolytic subclones and to characterize the individual genes responsible for the lipolytic activity. DNA sequence analysis of six genes revealed that the enzymes encoded by the metagenomic genes for lipolytic activity were novel with 34–48% similarity to known enzymes. They had conserved sequences similar to those in the hormone-sensitive lipase family. Based on their deduced amino acid similarity, the six genes encoding lipolytic enzymes were further divided into three subgroups, the identities among which ranged from 33% to 45%. The six predicted gene products were successfully expressed in E. coli and secreted into the culture broth. Most of the secreted enzymes showed a catalytic activity for hydrolysis of p-nitrophenyl butyrate (C₄) but not p-nitrophenyl palmitate (C₁₆).     

Mutational alteration of Bacillus subtilis DNA polymerase 3 to hydroxyphenylazopyrimidine resistance: polymerase 3 is necessary for DNA replication

A spontaneous mutant of $\text{Bacillus}\text{subtilis}$ resistant to killing by two hydroxyphenylazopyrimidines has been isolated. The DNA polymerase III of this mutant is resistant to inhibition by these drugs. The K_i for 6-(p-hydroxyphenylazo)-uracil (HPUra) is 20 μM, about 40 times higher than the K_i of the wild-type enzyme. The mutant and wild-type polymerases behave similarly during purification, are sensitive to N-ethylmaleimide and to 0.1 M KCl, and have the same K_m for dGTP (0.5 μM). The HPUra inhibition of both enzymes is attenuated competitively by dGTP. We conclude that polymerase III is the target for hydroxyphenylazopyrimidines $\text{in}\text{vivo}$, and since the drugs specifically inhibit replicative DNA synthesis, polymerase III is necessary for DNA replication.     

Genetic regulation of gibberellin deactivation in Pisum

The regulation of gibberellin (GA) deactivation was examined using the sin (slender) mutation in the garden pea (Pisum sativum L.). This mutation blocks the deactivation of GA₂₀, the precursor of the bioactive GA₁. Firstly, crosses were made to combine sin with the GA biosynthesis mutations na, lhⁱ and le-3. The combination sin na produced a novel phenotype, with long (‘slender’) basal internodes and extremely short (‘nana’) upper internodes. In contrast, the double mutant sin lhⁱ was phenotypically dwarf. The mutation sin causes an accumulation of GA₂₀ in maturing seeds, and this was unaffected by na, since the na mutation is not expressed in seeds. In contrast, lhⁱ seeds did not accumulate GA₂₀, since lhⁱ imposes an early block on GA biosynthesis. Secondly, the effects of sin on several steps in GA deactivation were investigated. In maturing seeds, the mutation sin blocks two steps in GA₂₀ metabolism, namely, GA₂₀ to GA₂₉, and GA₂₉ to GA₂₉-catabolite. In the vegetative plant, on the other hand, sin blocked the step GA₂₀ to GA₂₉, but not GA₂₉ to GA₂₉-catabolite; the steps GA₂₀ to GA₈₁ and GA₂₀ to GA₁ were also not impaired in this mutant. It is clear that the effects of sin, like those of na, are strongly organ-specific. The presence of separate enzymes for the steps GA₂₀ to GA₂₉ and GA₂₉ to GA₂₉-catabolite was suggested by the observation that GA₈ inhibited the latter step, but not the former, and by the inability of GA₂₀ and GA₂₉ to inhibit each other's metabolism. It is suggested that the Sin gene may be a regulatory gene controlling the expression of two structural genes involved in GA deactivation.     

The role of eucaryotic cell surface glycoconjugates in DNA replication

Multiple forms of ricin have been isolated from castor bean seeds. Two forms, ricin-1 and ricin-2, differ in their isoelectric pI values and toxicity towards IMR-32 cells. Inhibition of IMR-32 DNA polymerase α₂ is more pronounced with ricin-1 (65%) than with ricin-2 (10%). Ricin B chain (pI = 5.2) isolated from ricin-1 binds to IMR-32 cell surfaces as well as inhibits DNA polymerase α₂ activity when studiedin vitro. The presence of galβ-linked glycoconjugates near the active site of IMR-32 DNA polymerase α₂ has been proposed. Replication modulators which bind to the glycose portion of the enzymes involved in the replication system may need a mandatory binding to cell surface glycoconjugates for their activity.     

Catalytic properties of DNA polymerase α activity associated with the heat-stabilized nuclear matrix prepared from HeLa S3 cells

We have investigated whether or not ATP or other nucleoside di- and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase α associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37°C prior to subfractionation, as has been reported previously for DNA polymerase α bound to the nuclear matrix prepared from 22-h regenerating rat liver. We have found that HeLa cell matrix-associated DNA polymerase α activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase α activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix-bound DNA polymerase α activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2M NaCl or 0·25 M (NH₄)₂SO₄ and led us to consider that a 37° incubation of isolated nuclei renders resistant to high-salt extraction a form of DNA polymerase α which is unlikely to be involved in DNA replication in vivo.