Novel antiproliferative analogs of the Taq DNA polymerase inhibitor catalpol

The naturally occurring iridoid catalpol (1) is a Taq DNA polymerase inhibitor. However, its poor lipophilicity might account for the lack of biological activity against human solid tumor cell lines. The traditional prodrug approach by means of peracetylation of the free hydroxyl groups led to a compound, which showed a marginal growth inhibition against the most sensitive cell line A2780 (ovarian cancer). However, the formation of analogs bearing one to three silyl ether groups led to antiproliferative compounds against a panel of six human solid tumor cell lines, with GI50 values in the range 1.8-4.8 microM. Cell cycle studies revealed arrest in G0/G1 phase that is consistent with DNA polymerase inhibition.     

Inhibition of Taq DNA polymerase by seaweed extracts from British Columbia, Canada and Korea

Fifty-nine species of marine macrophytes from the coasts of British Columbia, Canada and Korea have been screened for the presence of PCR inhibitors, namely inhibitors of Taq DNA polymerase. Eleven of the species displayed some inhibitor activity. At the concentration of 5 μg of methanol extract in 25μL reaction mixture of PCR containing 1.5 unit of Taq DNA polymerase, one (Ulva sp.) of 8 Chlorophyta, eight (Colpomenia bullosa, Ecklonia cava, Endarachne binghamiae, Fucus distichus, Hizikia fusiformis, Sargassum confusum, Sargassum sagamianum, and Sargassum thunbergii) of 28 Phaeophyta, and one (Symphyocladia latiuscula) of 34 Rhodophyta showed inhibition in PCR amplification. In the case of the water extract, two (Cladophora columbiana, Ulva sp.) Chlorophyta, seven (Endarachne binghamiae, Fucus distichus, Hizikia fusiformis, Sargassum confusum, Sargassum sagamianum, Sargassum horneri, Scytosiphon dotyi) Phaeophyta, no Rhodophyta and one (Phyllospadix scouleri) seagrass showed inhibition in PCR amplification. the methanol fraction of Sargassum confusum and the water fraction of Fucus gardneri (mid–intertidal) have been found to inhibit PCR at level as low as 0.5 μg in 25μL of PCR reaction mixture. This revised version was published online in August 2006 with corrections to the Cover Date.     

Alternative dideoxy sequencing of double-stranded DNA by cyclic reactions using Taq polymerase.

The polymerase chain reaction (PCR) is a technique to amplify a specific DNA sequence millions of times. The thermostable enzyme Taq polymerase allows this procedure to take place under conditions of high specificity and automatization. By combining the techniques of PCR and dideoxy sequencing, it is possible to perform DNA sequencing independently of their structures. The cyclic sequencing reaction is carried out in the presence of an excess amount of sequencing primer and a radioactive nucleotide ([alpha-35S]dATP) using a DNA thermal cycler. Different reaction conditions were investigated and optimized including nucleotide ratios in each termination mix, primer/template ratios, amount of a radioactive nucleotide, and the program of the reaction. This method allows the detection of single base substitutions in heterozygous alleles, and the detection of homozygous deletions. A new RFLP of the human porphobilinogen deaminase (PBGD) gene was identified using this technique. This RFLP is created by one base difference (cytosine or adenine) that changes the restriction site for Apa LI. The alternative sequencing method described in this study is a simple and time-saving procedure that can also be used for large sequencing projects.     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling.

The possibility of increasing the affinity of a Taq DNA polymerase specific binding protein (affibody) was investigated by an alpha-helix shuffling strategy. The primary affibody was from a naive combinatorial library of the three-helix bundle Z domain derived from staphylococcal protein A. A hierarchical library was constructed through selective re-randomization of six amino acid positions in one of the two alpha-helices of the domain, making up the Taq DNA polymerase binding surface. After selections using monovalent phage display technology, second generation variants were identified having affinities (K(D)) for Taq DNA polymerase in the range of 30-50 nM as determined by biosensor technology. Analysis of binding data indicated that the increases in affinity were predominantly due to decreased dissociation rate kinetics. Interestingly, the affinities observed for the second generation Taq DNA polymerase specific affibodies are of similar strength as the affinity between the original protein A domain and the Fc domain of human immunoglobulin G. Further, the possibilities of increasing the apparent affinity through multimerization of affibodies was demonstrated for a dimeric version of one of the second generation affibodies, constructed by head-to-tail gene fusion. As compared with its monomeric counterpart, the binding to sensor chip immobilized Taq DNA polymerase was characterized by a threefold higher apparent affinity, due to slower off-rate kinetics. The results show that the binding specificity of the protein A domain can be re-directed to an entirely different target, without loss of binding strength.     

Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis

The polymerase chain reaction catalyzed by Taq DNA polymerase has been used for site-specific mutagenesis. The amplification was primed by two oligodeoxyribonucleotides complementary to insulin receptor cDNA. To direct the synthesis of mutant DNA, mismatches were introduced into one of the primers. Six different mutations were constructed by this technique. Of twelve clones whose sequences were determined, ten (83%) had the correct sequence. This technique, which does not require the use of single-stranded DNA templates, provides a simple and efficient approach to site-specific mutagenesis.     

Effects of agar and agarose on VentTM DNA polymerase and Taq DNA polymerase

Summary We have studied the effects of agar and agarose on Vent DNA polymerase and Taq DNA polymerase. Agar strongly inhibited Vent DNA polymerase but only moderately inhibited Taq DNA polymerase. Such a difference may be due to the fact that the two polymerases belong to different structural families. When Vent DNA polymerase is used to amplify DNA from lambda plaques, agarose rather than agar is the solid medium of choice.     

Dye structure affects Taq DNA polymerase terminator selectivity

All DNA sequencing methods have benefited from the development of new F667Y versions of Taq DNA polymerase. However, terminator chemistry methods show less uniform peak height patterns when compared to primer chemistry profiles suggesting that the dyes and/or their linker arms affect enzyme selectivity. We have measured elementary nucleotide rate and binding constants for representative rhodamine- and fluorescein-labeled terminators to determine how they interact with F667 versions of Taq Pol I. We have also developed a rapid gel-based selectivity assay that can be used to screen and to quantify dye-enzyme interactions with F667Y versions of the enzyme. Our results show that 6-TAMRA-ddTTP behaves like unlabeled ddTTP, while 6-FAM-ddTTP shows a 40-fold reduction in the rate constant for polymerization without affecting ground-state nucleotide binding. Detailed mechanism studies indicate that both isomers of different fluorescein dyes interfere with a conformational change step which the polymerase undergoes following nucleotide binding but only when these dyes are attached to pyrimidines. When these same dyes are attached to purines by the same propargylamino linker arm, they show no effect on enzyme selectivity. These studies suggest that it may be possible to develop fluorescein terminators for thermocycle DNA sequencing methods for polymerases that do not discriminate between deoxy- and dideoxynucleotides.     

Inhibition of DNA polymerase by captan

Captan inhibits DNA polymerases of both eukaryotic and prokaryotic sources. When polymerases were employed in assays with various polynucleotides as template-primer, no specificity in the base sequence of polynucleotide was required for inhibition. Sucrose gradient centrifugation and preincubation studies showed the inhibition was caused by an irreversible alteration of the polymerase. Captan and DNA compete for the same site on the polymerase, thus DNA can serve a protective role in the elimination of captan's action. The pyrophosphate exchange activity associated with the polymerase is not inhibited by captan and the fidelity with which DNA polymerase I copies the DNA template also is not altered by captan treatment.     

Inhibition of RNA-directed DNA polymerase by aurintricarboxylic acid.

Commercial-grade aurintricarboxylic acid (ATA) inhibits poly(A), poly(C) and viral RNA-directed DNA synthesis by detergent-disrupted virions of Moloney murine leukemia virus. Paper chromatography of crude ATA yields two active components, which appear to behave identically, and at least two inactive components. The concentration of ATA needed to inhibit polymerase activity is proportional to the concentration of viral protein. The inhibition is neither attributable to contaminating heavy metal ions in the ATA preparation nor to chelation by ATA of Mn2+ or Zn2+, the necessary co-factors. Inhibition of the polymerase reaction by ATA greatly increases the Km for the primer [oligo(T)/oligo(dG)], while it only slightly lowers the Vmax and does not affect the Km's for the template [poly(A)/poly(C)] or the substrate (TTP/dGTP). Thus, ATA seems to reduce specifically the affinity of the polymerase for the DNA primer molecule.     

Inhibition of DNA polymerase alpha by aphidicolin derivatives.

17-Acetylaphidicolin was 10-fold weaker and two derivatives lacking hydroxyl groups at the 16 and 17 positions were 100-fold weaker than aphidicolin as inhibitors of DNA polymerase alpha from HeLa and Chinese hamster ovary cells. 17,18-Diacetyl, 3,17,18-triacetyl and 3-epi derivatives of aphidicolin were inactive. Active compounds were, like aphidicolin, competitive with dCTP and did not inhibit aphidicolin-resistant DNA polymerases.     

Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase.

A polymerase stop assay has been developed to determine the DNA nucleotide sequence specificity of covalent modification by antineoplastic agents using the thermostable DNA polymerase from Thermus aquaticus and synthetic labelled primers. The products of linear amplification are run on sequencing gels to reveal the sites of covalent drug binding. The method has been studied in detail for a number of agents including nitrogen mustards, platinum analogues and mitomycin C, and the sequence specificities obtained accord with those obtained by other procedures. The assay is advantageous in that it is not limited to a single type of DNA lesion (as in the piperidine cleavage assay for guanine-N7 alkylation), does not require a strand breakage step, and is more sensitive than other primer extension procedures which have only one cycle of polymerization. In particular the method has considerable potential for examining the sequence selectivity of damage and repair in single copy gene sequences in genomic DNA from cells.     

Inhibition of DNA polymerase activity by thymine hydrates.

Ultraviolet irradiation of DNA results in various pyrimidine modifications. We have demonstrated formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in UV-irradiated poly(dA-dT):poly(dA-dT). Both are released from DNA as free bases by bacterial and human glycosylases. Thymine hydrates are stable in DNA and can be detected in control, unirradiated substrates. We examined the effects of thymine hydrates in UV-irradiated substrate poly(dA-dT):poly(dA-dT) on E. coli DNA polymerase I activity. Enzymic incorporation of labeled thymidine-5'-monophosphate significantly decreased with increasing UV dose. Reversal of DNA thymine hydrates to thymines by mild heating of the substrate prior to enzymic reaction resulted in partial recovery of nucleotide incorporation. Cyclobutane thymine dimers are formed between non-adjacent thymines in UV-irradiated poly(dA-dT):poly(dA-dT). These are responsible for the incomplete recovery of DNA polymerase activity following heating due to their heat stability. Analyses of the irradiated and hydrolyzed substrate also demonstrated formation of minor yields of photoproducts formed by covalent linkage of adjacent thymines and adenines by UV-irradiation. Therefore, the thymine hydrates formed in UV-irradiated DNA partially inhibit polymerase activity during DNA synthesis and thus could be potentially lethal if unrepaired.     

Development of a new DNA sequencing method: 3'-ester cleavage catalyzed by Taq DNA polymerase.

A new 3'-esterified dTTP is incorporated into DNA by Taq DNA polymerase but does not act as a chain terminator. The esterase activity of the polymerase seems to be template dependent and occurs only if the next correct nucleotide is present.