Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate whether DNA replication in malignant cells deviates from that of normal cells we compared DNA polymerases alpha, delta, and epsilon from normal rat liver to the enzymes from fast-growing (malignant) Novikoff hepatoma cells. DNA polymerases were purified 300-fold by three chromatographic steps. Characterization included measurement of physicochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), quantitation of catalytic activities using specific DNA primer templates (Km values) and inhibitors (Ki values), and identification of polypeptides which are strongly associated with DNA polymerases. Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. In contrast, the DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with the specific primer templates gapped calf thymus DNA and poly(dA.dT), were higher. Furthermore, sedimentation and diffusion coefficients, Stokes' radii, and frictional coefficient ratios of DNA polymerases alpha and epsilon from malignant cells significantly deviated. In addition, when the dNTP-binding sites were probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP), significantly lower Ki values were obtained for the polymerases from Novikoff cells indicating lower affinity of the dNTP binding site to deoxyribonucleoside 5'-triphosphates. Altered catalytic and molecular properties are possibly a consequence of malignant transformation. It is to be expected that similar changes occur in DNA polymerases of other tumors. In particular, diminished affinity to primer templates and weakened nucleotide binding leads to lowered specificity of nucleotide selection in the base-pairing process and is therefore likely to cause an enhanced mutation rate during malignant progression.
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PMID:DNA polymerases alpha, delta, and epsilon of Novikoff hepatoma cells differ from those of normal rat liver in physicochemical and catalytic properties. 767 Sep 30

A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented.
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PMID:NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides. 772 21

DNA templates with 8-hydroxyguanine (7,8-dihydro-8-oxoguanine, oh8Gua) at a site corresponding to the first or second position of codon 12 of the c-Ha-ras gene were prepared, and the nucleotides inserted opposite the modified base were compared. The Klenow fragment (KF) of Escherichia coli DNA polymerase I inserted C opposite oh8Gua at both positions. Taq DNA polymerase incorporated C and A opposite oh8Gua, and the ratio of C to A was higher at the first position than at the second position. DNA polymerase alpha (pol alpha) inserted A and C at the first position, and A at the second position of codon 12, indicating that the ratio of C to A was higher at the first position. Moreover, we studied the extensions of bases paired with oh8Gua by DNA polymerases with or without 3'-5' exonuclease activity. G and T opposite oh8Gua were removed, and subsequently C was inserted by KF. We found that an oh8Gua:A pair was recognized by the exonuclease activity of the enzyme and that A was partially substituted by C. On the other hand, pol alpha extended only C and A opposite oh8Gua. No difference was observed with oh8Gua at the two positions. These results indicate that the ratio of nucleotides incorporated opposite oh8Gua depends on the sequence context, while there is no particular difference in the extension of base pairs involving oh8Gua by DNA polymerases.
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PMID:Comparison of incorporation and extension of nucleotides in vitro opposite 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in hot spots of the c-Ha-ras gene. 774 97

It has previously been suggested that inhibition of the proofreading 3'-5' exonuclease activity of DNA polymerase may play an important role in generation of UV-induced mutations in Escherichia coli. Our previous work showing that overproduction of epsilon, the proofreading subunit of DNA polymerase III, counteracts the SOS mutagenic response of E. coli seemed to be consistent with this hypothesis. To explore further the nature of the antimutagenic effect of epsilon we constructed plasmid pMK17, which encodes only two of the three highly conserved segments of epsilon--ExoI and ExoII; the third segment, ExoIII, which is essential for 3'-5' exonuclease activity, is deleted. We show that at 40 degrees C, overproduction of the truncated epsilon subunit significantly delays production of M13 phage, suggesting that the protein retains its capacity to bind to DNA. On the other hand, the presence of pMK17 in a trpE65 strain growing at 40 degrees C causes a 10-fold decrease in the frequency of UV-induced Trp+ mutations. This antimutagenic effect of the truncated epsilon is effectively relieved by excess UmuD,C proteins. We also show that the presence of plasmid pIP21, which contains the dnaQ49 allele encoding an epsilon subunit that is defective in proofreading activity, almost completely prevents generation of UV-induced mutations in the trpE65 strain. We propose that the DNA binding ability of free epsilon, rather than its 3'-5' exonuclease activity, affects processing of premutagenic UV-induced lesions, possibly by interfering with the interaction between the UmuC-UmuD'-RecA complex and Pol III holoenzyme. This interaction is probably a necessary condition for translesion synthesis.
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PMID:The antimutagenic effect of a truncated epsilon subunit of DNA polymerase III in Escherichia coli cells irradiated with UV light. 775 31

Reductively-activated mitomycin C (MC) presents a high specificity to the 5'-CG site and to a lesser extent the 5'-GG site. However, its affinity is different for each 5'-CG site. This was evidenced by using the 3'-5' exonuclease activity of T4 DNA polymerase on a short DNA fragment exposed to MC, which was gradually activated by several Na2S2O4 additions. The time-delayed appearance of some exonuclease digestion stop sites (corresponding to MC-monofunctional adducts) suggests that MC discriminates between very fine structural variations. The feature of the stop sites suggests a good fit of MC in the DNA groove, in the case of the major alkylation sites, but not in the case of a minor 5'-TG alkylation site. Furthermore, it is evidenced by the use of the chemical probe hydroxylamine (HA) that MC-monoalkylation of 5'-CG (or 5'-GG) does not induce notable local structural disturbance of the DNA double helix, as opposed to alkylation of the 5'-TG site of minor specificity, which leads to significant local DNA distortion. This suggests that the 'in vivo' effect of MC is related, not only to amount of alkylated sites (essentially 5'-CG sites), but also to possible local DNA deformations (at minor alkylation sites).
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PMID:Mitomycin C-induced distortions of DNA at minor alkylation sites. 782 Aug 85

phi 29 DNA polymerase shares with other DNA-dependent DNA polymerases several regions of amino acid homology along the primary structure. A conserved amino acid motif, located in the C-terminal portion of the polypeptide and characterized by the amino acid sequence KK(K/R)Y, is conserved in the group of eukaryotic-type DNA polymerases. In the subgroup of DNA polymerases that have a protein-priming mechanism, this motif is restricted to the sequence KXY, X never being a positively charged amino acid. Residues Lys498 and Tyr500 form this conserved motif in phi 29 DNA polymerase. Mutant K498T, in which the positive charge of the motif has been eliminated, was strongly affected both in initiation (terminal protein-dAMP formation, using terminal protein as primer) and DNA polymerization reactions. Mutants K498R and Y500S were able to carry out the initiation reaction to a higher or similar extent, respectively, than wild-type phi 29 DNA polymerase but were affected in DNA polymerization reactions. All of the mutations severely affected the stable binding of the polymerase to a primer-template DNA. In addition, all of the mutant polymerases analyzed in this work showed an unusually strong 3'-5' exonuclease activity both under polymerization or non-polymerization conditions. The results obtained suggest a role of the conserved residues of the KXY motif in stabilizing the primer terminus at the polymerization active site, the positive charge of residue Lys498 being critical for the synthetic activities of phi 29 DNA polymerase.
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PMID:Primer terminus stabilization at the phi 29 DNA polymerase active site. Mutational analysis of conserved motif KXY. 785 44

The in vitro replication of synthetic oligodeoxyribonucleotides carrying internucleotide polyphosphate groups or alkanediol "spacers" of various sizes with the use of various DNA polymerases has been studied. All modifications, except for the diphosphate group, almost completely block the polymerization process. In the case of AMV reverse transcriptase, Taq and T7 DNA polymerases and also the Klenow fragment of E. coli DNA polymerase I, a template-independent addition of a nucleotide at the 3' end of the incomplete replica was observed. T4 DNA polymerase, displaying the strongest 3'-5' exonuclease activity among the polymerases studied, did not incorporate additional nucleotides. The use of oligonucleotides with non-nucleotide inserts as primers in polymerase chain reaction (PCR) allows to obtain DNA copies with protruding 5'-termini, suitable for hybridisation analysis.
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PMID:[Features of replicating synthetic oligonucleotides with non-native chains]. 788 Jan 80

Accurate synthesis of DNA by polymerase is due in part to the selective removal of misincorporated nucleotides by a 3'-5' exonuclease activity (proofreading). Proofreading by an exonuclease domain containing a single-stranded DNA binding site may involve local melting of a duplex DNA substrate. Here we use time-resolved fluorescence spectroscopy to analyze the local melting of a DNA duplex terminus induced by the Klenow fragment of DNA polymerase I. Four oligodeoxynucleotide primer/templates were prepared, each containing the fluorescent adenine analog 2-aminopurine (A*) at the primer 3' terminus, and one of the common DNA bases opposite the A* residue. Fluorescence decays of the duplex DNAs and the single primer oligonucleotide were jointly analyzed using global analysis procedures. Four lifetime components were resolved in the duplex DNAs, representing distinct conformational states of the terminal A* residue: paired A* bases, partially stacked A* bases, and extended A* bases. The variation of the apparent fraction of paired A* bases with temperature was in accord with optical melting data, and the extent of base pairing observed in each duplex was consistent with the base-pairing preferences of A* established in other studies. These results establish that the fluorescence decay characteristics of A* can be used to examine base-pairing interactions at a DNA duplex terminus. Since the fluorescence of A* can be observed without interference from protein amino acid residues, unlike existing methods for monitoring DNA melting transitions, this method was used to examine the extent to which Klenow fragment could induce fraying at each duplex terminus.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Melting of a DNA helix terminus within the active site of a DNA polymerase. 791 16

Fluorescence depolarization decays were measured for 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) probes attached internally to 17-mer.27-mer oligonucleotides bound to Klenow fragment of DNA polymerase I. The time-resolved motions of the dansyl probes were sensitive indicators of DNA-protein contacts, showing that the protein binds to DNA with two footprints, corresponding to primer termini at either the polymerase or 3'-5' exonuclease sites. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3- to 4-fold increase in the equilibrium partitioning of DNA into the exonuclease site; the largest effects were observed for purine-purine mismatches. Two or more consecutive G.G mismatches caused the DNA to bind exclusively at the exonuclease site, with a partitioning constant at least 250-fold greater than that of the corresponding matched DNA sequence. Internal single mismatches produced larger effects than the same mismatch at the primer terminus, with a delta delta G relative to the matched sequence of -1.1 to -1.3 kcal/mol for mismatches located 2, 3, or 4 bases from the primer terminus. Although part of the observed effects may be attributed to the increased melting capacity of the DNA, it appears that the polymerase site also promotes movement of DNA into the exonuclease site by rejecting aberrant primer termini. These effects suggest that the polymerase and exonuclease sites act together to recognize specific errors that distort the primer terminus, such as frameshifts, in addition to proofreading misincorporated bases.
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PMID:Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I. 793 11

We have overexpressed the vaccinia virus DNA polymerase using the hybrid vaccinia virus/T7 expression system. Accumulation of the DNA polymerase to levels as high as 10% of the total protein was observed following coinfection of BSC40 cells with the appropriate vaccinia recombinants. Although the DNA polymerase produced at 37 degrees C was largely insoluble, 25% of the recombinant protein could be recovered as soluble protein when infected cultures were maintained at 32 degrees C. Starting with cytoplasmic lysates of coinfected cells, a rapid and reproducible purification protocol that yielded apparently homogeneous preparations of the DNA polymerase after four chromatographic steps was established. Typically, 0.3 mg of purified DNA polymerase was obtained from 27 mg of total protein within 10 h after harvesting infected cells. As was previously described for the DNA polymerase purified from vaccinia-infected cells (Challberg and Englund, J. Biol. Chem., 254, 7812-7819, 1979), the purified recombinant enzyme displayed both polymerase and 3'-5' exonuclease activities but lacked detectable 5'-3' exonuclease activity. Kinetic analysis of nucleotide incorporation catalyzed by the vaccinia enzyme revealed apparent Km values of 0.9, 2.9, 4.0, and 2.7 microM for dGTP, dATP, TTP, and dCTP, respectively.
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PMID:Overexpression and purification of the vaccinia virus DNA polymerase. 795 Mar 89


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