Mechanism of Inhibition of DNA Synthesis by 1-β-D-Arabinofuranosyl-E-5-(2-Bromovinyl)uracil

The mechanism of the inhibitory action of 1-β-D -arabinofuranosyl-E-5-(2-bromovinyl) uracil triphosphate (BV-araUTP) on DNA synthesis by Escherichia coli DNA polymerase I Klenow fragment was studied. Acting as a chain terminator, BV-araUTP inhibited DNA synthesis by Klenow fragment more effectively than 2′, 3′-dideoxythymidine triphosphate (ddTTP). However, the incorporation sites of BV-araU monophosphate were restricted at consecutive dTMP sequence whereas ddTMP was incorporated at every dTMP site.     

Pα-Methyl Deoxynucleoside Triphosphates as Substrates for E. COLI DNA Polymerase I in a Template-Directed Synthesis of DNA

Abstract

Pα-methyl deoxynucleoside triphosphates are used as substrates for E. coli DNA polymerase I in template-directed polymerase reactions. It is shown that the modified compounds are incorporated together with the unmodified deoxynucleoside triphosphates into DNA under both nick-translation and Klenow reaction conditions.     

Detection of enzymatic activities in sodium dodecyl sulfate-polyacrylamide gels: DNA polymerases as model enzymes

Recent techniques for detecting the catalytic activity of enzymes in sodium dodecyl sulfate (SDS)-polyacrylamide gels have been hampered by lack of reproducibility associated with variability in commercial SDS preparations. Simple expedients which facilitate reproducible detection of DNA polymerase activity and which appear to be widely applicable to detection of other enzymes are reported here. It was observed that reproducibility of a reported procedure for DNA polymerase detection (Spanos, A., Sedgwick, S. G., Yarranton, G. T., Hübscher, U., and Banks, G. R. (1981) Nucl. Acids Res.9, 1825–1839) depends on the SDS used for electrophoresis, and that sensitivity is markedly reduced if currently available SDS is substituted for the discontinued product specified by Spanos et al. A modified procedure yielding sensitivity with contemporary commercial SDS, which exceeds the sensitivity observed when using the protocol and the SDS originally specified, is described. The modifications employed, which presumably promote renaturation of enzymes, are (1) embedding fibrinogen in gels and (2) washing detergent from gels with aqueous isopropanol after electrophoresis. These expedients permit detection of picogram amounts of Escherichia coli DNA polymerase I and its Klenow fragment and nanogram amounts of calf thymus α and rat liver (Novikoff hepatoma) β polymerases. Finally, it is shown that sensitivity of DNA polymerase detection is reduced by lipophilic contaminants in contemporary commercial SDS, and that the expedients employed here mitigate the deleterious effect of these impurities.     

Exploration of factors driving incorporation of unnatural dNTPS into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase alpha

In order to further understand how DNA polymerases discriminate against incorrect dNTPs, we synthesized two sets of dNTP analogues and tested them as substrates for DNA polymerase α (pol α) and Klenow fragment (exo⁻) of DNA polymerase I (Escherichia coli). One set of analogues was designed to test the importance of the electronic nature of the base. The bases consisted of a benzimidazole ring with one or two exocyclic substituent(s) that are either electron-donating (methyl and methoxy) or electron-withdrawing (trifluoromethyl and dinitro). Both pol α and Klenow fragment exhibit a remarkable inability to discriminate against these analogues as compared to their ability to discriminate against incorrect natural dNTPs. Neither polymerase shows any distinct electronic or steric preferences for analogue incorporation. The other set of analogues, designed to examine the importance of hydrophobicity in dNTP incorporation, consists of a set of four regioisomers of trifluoromethyl benzimidazole. Whereas pol α and Klenow fragment exhibited minimal discrimination against the 5- and 6-regioisomers, they discriminated much more effectively against the 4- and 7-regioisomers. Since all four of these analogues will have similar hydrophobicity and stacking ability, these data indicate that hydrophobicity and stacking ability alone cannot account for the inability of pol α and Klenow fragment to discriminate against unnatural bases. After incorporation, however, both sets of analogues were not efficiently elongated. These results suggest that factors other than hydrophobicity, sterics and electronics govern the incorporation of dNTPs into DNA by pol α and Klenow fragment.     

Display of functional nucleic acid polymerase on Escherichia coli surface and its application in directed polymerase evolution

We report a first of its kind functional cell surface display of nucleic acid polymerase and its directed evolution to efficiently incorporate 2′-O-methyl nucleotide triphosphates (2′-OMe-NTPs). In the development of polymerase cell surface display, two autotransporter proteins (Escherichia coli adhesin involved in diffuse adherence and Pseudomonas aeruginosa esterase A [EstA]) were employed to transport and anchor the 68-kDa Klenow fragment (KF) of E. coli DNA polymerase I on the surface of E. coli. The localization and function of the displayed KF were verified by analysis of cell outer membrane fractions, immunostaining, and fluorometric detection of synthesized DNA products. The EstA cell surface display system was applied to evolve KF for the incorporation of 2′-OMe-NTPs and a KF variant with a 50.7-fold increased ability to successively incorporate 2′-OMe-NTPs was discovered. Expanding the scope of cell-surface displayable proteins to the realm of polymerases provides a novel screening tool for tailoring polymerases to diverse application demands in a polymerase chain reaction and sequencing-based biotechnological and medical applications. Especially, cell surface display enables novel polymerase screening strategies in which the heat-lysis step is bypassed and thus allows the screening of mesophilic polymerases with broad application potentials ranging from diagnostics and DNA sequencing to replication of synthetic genetic polymers.     

T7 DNA polymerase in automated dideoxy sequencing.

T7 DNA polymerase with chemically inactivated 3'-5' exo-nuclease activity, as well as unmodified T7 DNA polymerase, were used for sequencing by the dideoxy method in an automated system with fluorescence labelled primer and on-line detection of laser-excited reaction products. An analysis of signal intensity variations in the C track revealed that low C signals were usually preceded by a T in the sequence. This effect was modified by surrounding nucleotides. Signal intensities were more uniform with T7 polymerase than with the Klenow fragment of DNA polymerase I. Some sequences ambiguous with the Klenow enzyme could easily be evaluated with the T7 enzyme. One sequence could only be read by the unmodified T7 polymerase, while both the Klenow fragment and the chemically modified T7 enzyme gave uninterpretable data.     

Behavior of the large fragment of DNA polymerase I (the Klenow fragment) during fractionation of a cell-free extract of E. coli MRE-600

Distribution of the DNA polymerase I large fragment (Klenow fragment) was studied during fractionation of the E. coli MRE-600 cell-free extract with polyethylenimine. On the basis of the results obtained a simple procedure is proposed that enables the Klenow fragment to be obtained as a coproduct of DNA polymerase I, RNA polymerase, polynucleotide phosphorylase, nucleotide kinases with acetokinase and nucleoside deoxy-ribosyltransferase in the framework of a combined technological scheme.     

Sufficiency of the Klenow fragment for survival of polC(Ts) pcbA1 Escherichia coli at 43 degrees C.

Escherichia coli cells with the pcbA1 allele and temperature-sensitive DNA polymerase III mutations survived at a restrictive temperature if DNA polymerase I activity was present. The Klenow fragment of DNA polymerase I was capable of supporting cell survival as well.     

Gold nanoparticles-based colorimetric assay for rapid detection of Salmonella species in food samples

A rapid, sensitive and cost-effective method was developed for detection of foodborne pathogens, particularly Salmonella species. The method utilizes single stranded DNA (ssDNA) probes and non-functionalized gold nanoparticles to provide a colorimetric assay for the detection of PCR amplified DNA. Different food samples were tested with the PCR-based colorimetric assay parallel with the conventional culture method. The sensitivity and specificity of colorimetric assay was 89.15 and 99.04% respectively with reference to conventional culture method. The total time required to detect the Salmonella spp. present in food samples by the developed method is less than 8 h, including 6 h incubation. It was observed that the colorimetric assay was 10 times more sensitive than gel-based detection with the same concentration of DNA used for analysis.     

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     

Interactions of replication versus repair DNA substrates with the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus

Different DNA polymerases partition differently between replication and repair pathways. In this study we examine if two Pol I family polymerases from evolutionarily distant organisms also differ in their preferences for replication versus repair substrates. The DNA binding preferences of Klenow and Klentaq DNA polymerases, from Escherichia coli and Thermus aquaticus respectively, have been studied using a fluorescence competition binding assay. Klenow polymerase binds primed-template DNA (the replication substrate) with up to 50× higher affinity than it binds to nicked DNA, DNA with a 2 base single-stranded gap, blunt-ended DNA, or to a DNA end with a 3′ overhang. In contrast, Klentaq binds all of these DNAs almost identically, indicating that Klenow has a stronger ability to discriminate between replication and repair substrates than Klentaq. In contrast, both polymerases bind mismatched primed-template and blunt-ended DNA tighter than they bind matched primed-template DNA, suggesting that these two proteins may share a similar mechanism to identify mismatched DNA, despite the fact that Klentaq has no proofreading ability. In addition, the presence or absence of 5′- or 3′-phosphates has slightly different effects on DNA binding by the two polymerases, but again reinforce Klenow's more effective substrate discrimination capability.     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase      

Formamide as a denaturant for bisulfite conversion of genomic DNA: Bisulfite sequencing of the GSTPi and RARβ2 genes of 43 formalin-fixed paraffin-embedded prostate cancer specimens

Analysis of methylated DNA, which refers to 5-methycytosine (5mC) versus cytosine (C) at specific loci in genomic DNA (gDNA), has received increased attention in epigenomics, particularly in the area of cancer biomarkers. Many different methods for analysis of methylated DNA rely on initial reaction of gDNA with concentrated acidic sodium bisulfite to quantitatively convert C to uracil (U) via sulfonation of denatured, single-stranded gDNA under conditions where 5mC is resistant to analogous sulfonation leading to thymine (T). These methods typically employ polymerase chain reaction (PCR) amplification after bisulfite conversion, thereby leading to readily detectable amounts of amplicons where T and C are measured as surrogates for C and 5mC in the original unconverted gDNA. However, incomplete bisulfite conversion of C in gDNA has been reported to be a common source of error in analysis of methylated DNA. Incomplete conversion can be revealed during the course of bisulfite sequencing, which is the generally accepted “gold standard” for analysis of methylated DNA. Previous bisulfite sequencing investigations of conventional predenaturation of gDNA with NaOH followed by the use of bisulfite containing added urea to maintain denaturation and thus mitigate incomplete conversion of C have been reported to give conflicting results. The current study describes a new approach where conventional predenaturation of gDNA with NaOH is instead achieved with formamide and maintains denaturation during subsequent sample handling and sulfonation. This formamide-based method was applied to 46 formalin-fixed/paraffin-embedded (FFPE) biopsy tissue specimens from well-characterized patients with primary prostate cancer. These specimens were representative of difficult-to-analyze samples due to the chemically compromised nature of the gDNA, which was recovered by modifying the protocol for a commercially available total RNA/DNA extraction kit (RecoverALL). An additional novel aspect of this study was analysis of CpG-rich promoter regions of two prostate cancer-related genes: glutathione S-transferase pi (GSTPi) and retinoic acid receptor beta2 (RARβ2). High-quality bisulfite sequencing results were obtained for both genes in 43 of 46 (93%) specimens. Detection of methylated GSTPi and RARβ2 genes was significantly associated with primary prostate cancer as compared with the benign prostate (Fisher’s exact test, P < 0.001). The sensitivity and specificity of detection of methylated GSTPi and RARβ2 genes were 86% and 100% and 91% and 100%, respectively. Moreover, the presence of either methylated gene was detected in primary prostate cancer with sensitivity and specificity of 100% and 100%, respectively. The results demonstrated a high degree of reliability of formamide-based denaturation and bisulfite conversion that should extend, generally, to FFPE and other types of samples intended for any analytical method predicated on bisulfite conversion. This pilot study also demonstrated the efficacy of determining methylation of these two genes with high sensitivity and specificity in FFPE biopsy tissue specimens. Moreover, the results showed a highly significant association of methylated GSTPi and RARβ2 genes with primary prostate cancer. Finally, this improved procedure for determining these two methylated genes may allow the detection of prostate cancer cells in core biopsy specimens with insufficient numbers of cells and poor morphology.     

Oligonucleotides labeled with single fluorophores as sensors for deoxynucleotide triphosphate binding by DNA polymerases

Oligonucleotides labeled with a single fluorophore (fluorescein or tetramethylrhodamine) have been used previously as fluorogenic substrates for a number of DNA modifying enzymes. Here, it is shown that such molecules can be used as fluorogenic probes to detect the template-dependent binding of deoxynucleotide triphosphates by DNA polymerases. Two polymerases were used in this work: the Klenow fragment of the Escherichia coli DNA polymerase I and the Bacillus stearothermophilus polymerase, Bst. When complexes of these polymerases with dye-labeled hairpin-type oligonucleotides were mixed with various deoxynucleotide triphosphates in the presence of Sr²⁺ as the divalent metal cation, the formation of ternary DNA-polymerase-dNTP complexes was detected by concentration-dependent changes in the fluorescence intensities of the dyes. Fluorescein- and tetramethylrhodamine-labeled probes of identical sequences responded differently to the two polymerases. With Bst polymerase, the fluorescence intensities of all probes increased with the next correct dNTP; with Klenow polymerase, tetramethylrhodamine-labeled probes increased their fluorescence, but the intensity of fluorescein-labeled probes decreased on formation of ternary complexes with the correct incoming nucleotides. The use of Sr²⁺ as the divalent metal ion allowed the formation of catalytically inactive ternary complexes and obviated the need for using 2′,3′-dideoxy-terminated oligonucleotides as would have been needed in the case of Mg²⁺ as the metal ion.     

Family A and family B DNA polymerases are structurally related: evolutionary implications.

In order to establish the evolutionary relationship between the family A and B DNA polymerases, we have closely compared the 3'-->5' exonuclease domains between the Klenow fragment of E.coli DNA polymerase I (a family A DNA polymerase) and the bacteriophage PRD1 DNA polymerase, the smallest member of the DNA polymerase family B. Although the PRD1 DNA polymerase has a smaller 3'-->5' exonuclease domain, its active sites appear to be very similar to those of the Klenow fragment. Site-directed mutagenesis studies revealed that the residues important for the 3'-->5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in the PRD1 DNA polymerase as well. The metal binding ligands are also essential for the strand-displacement activity of the PRD1 DNA polymerase. Based on these results and the studies by others in various systems, we conclude that family A and B DNA polymerases, at least in the 3'-->5' exonuclease domain, are structurally as well as evolutionarily related.     

Comparison of DIG-11-dUTP utilization by Geobacillus caldoxylosilyticus TK4, Mycobacterium tuberculosis and Escherichia coli DNA polymerases

DNA polymerases are used for many applications and we comparatively investigated DNA synthesis activity of DNA polymerase I enzymes of Geobacillus caldoxylosilyticus TK4, Escherichia coli and Mycobacterium tuberculosis with DIG-11-dUTP using synthetic DNA substrates. We showed that Gca polymerase I and Klenow Fragment (KF) used DIG-11-dUTP instead of dTTP almost at the same ratio, but more efficiently than Mtb polymerase I. We considered that Gca polymerase I could be efficiently used to label a DNA oligonucleotide either internally or at the 3′-terminus by DIG-11-dUTP for the generation of non-radioactive labeled DNA substrates at higher temperature than KF. All three polymerases could not elongate the primer terminus after adding ddNTPs into DNA that is characteristic for all known DNA polymerase I enzymes.