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)

Enzyme kinetic measurements are presented showing that Km rather than maximum velocity (Vmax) discrimination governs the frequency of forming 2-aminopurine X cytosine base mispairs by DNA polymerase alpha. An in vitro system is used in which incorporation of dTMP or dCMP occurs opposite a template 2-aminopurine, and values for Km and Vmax are obtained. Results from a previous study in which dTTP and dCTP were competing simultaneously for insertion opposite 2-aminopurine indicated that dTMP is inserted 22 times more frequently than dCMP. We now report that the ratio of Km values KCm/KTm = 25 +/- 6, which agrees quantitatively with the dTMP/dCMP incorporation ratio obtained previously. We also report that VCmax is indistinguishable from VTmax. These Km and Vmax data are consistent with predictions from a model, the Km discrimination model, in which replication fidelity is determined by free energy differences between matched and mismatched base pairs. Central to this model is the prediction that the ratio of Km values for insertion of correct and incorrect nucleotides specifies the insertion fidelity, and the maximum velocities of insertion are the same for both nucleotides.
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PMID:Kinetic measurement of 2-aminopurine X cytosine and 2-aminopurine X thymine base pairs as a test of DNA polymerase fidelity mechanisms. 695 28

[3H]2-Aminopurine deoxyribonucleoside triphosphate and [32P]dATP were added exogenously at equimolar concentrations to washed HeLa cell nuclei both in the presence and absence of cell cytoplasm. The observed ratio of 2-aminopurine/adenine deoxyribonucleotide incorporation into DNA was about 12%, which is consistent with 2-aminopurine misinsertion frequencies measured in cell-free assays, for various DNA polymerases including alpha-polymerase from calf thymus, Escherichia coli polymerase I, and several mutant and wild type bacteriophage T4 polymerases. Based on the 12% 2-aminopurine/adenine misincorporation ratio, we propose that proofreading of replicating DNA is not occurring in HeLa nuclei, and that discrimination against 2-aminopurine incorporation is governed primarily by a 1.1 kcal/mol difference in free energy between 2-aminopurine.thymine and adenine.thymine base pairs rather than by properties attributable to either the mammalian DNA polymerase or HeLa cell nuclear replication apparatus.
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PMID:Evidence for the absence of DNA proofreading in HeLa cell nuclei. 725 86

The fluorescent properties and their sensitivity to the surrounding environment of the nucleotide analog 2-aminopurine (2-AP) have been well documented. In this paper we describe the use of 2-AP as a direct spectroscopic probe of the mechanism of nucleotide incorporation by Escherichia coli Pol I Klenow fragment (KF) and bacteriophage T4 DNA polymerase. The nucleotidyl transfer reaction may be monitored in real time by following the fluorescence of 2-AP, allowing the detection of transient intermediates along the reaction pathway that are inaccessible through traditional radioactive assays. Previous studies with Klenow fragment [Kuchta, R. D., Mizrahi, V., Benkovic, P. A., Johnson, K. A., & Benkovic, S. J. (1987) Biochemistry 26, 8410-8417] have revealed the presence of a nonchemical step prior to chemistry and have identified this conformational change as the rate-limiting step of correct nucleotide incorporation. During correct incorporation, phosphodiester bond formation occurs at a rate greater than the conformational change and has not been measured. However, during misinsertion, the rate of the chemical step becomes partially rate limiting and it becomes possible to detect both steps. We have successfully decoupled the chemical and conformational change steps for nucleotide insertion by KF using the misincorporation reaction, and we present direct spectroscopic evidence for an activated KF'-DNA-dNTP species following the conformational change step which features hydrogen bonding between the incoming and template bases. In addition, we have utilized these same experiments to demonstrate the existence of a similar nonchemical step in the mechanism of dNTP incorporation by bacteriophage T4 DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The nucleotide analog 2-aminopurine as a spectroscopic probe of nucleotide incorporation by the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase. 761 19

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

The effects of local DNA sequence on the proofreading efficiency of wild-type T4 DNA polymerase were examined by measuring the kinetics of removal of the fluorescent nucleotide analog 2-aminopurine deoxynucleoside monophosphate (dAPMP) from primer/templates of defined sequences. The effects of (1) interactions with the 5'-neighboring bases, (2) base pair stability, and (3) G.C content of the surrounding sequences on the pre-steady-state kinetics of dAPMP excision were measured. Rates of excision dAPMP from a primer 3'-terminus located opposite a template T (AP.T base pair) increased, over a 3-fold range, with the 5'-neighbor to AP in the order C < G < T < A. Rates of removal of dAPMP from AP.X base pairs located in the same surrounding sequence increased as AP.T < AP.A < AP.C < AP.G, which correlates with the decrease in the stabilities of these base pairs predicted by Tm measurements. A key finding was that AP was excised at a slower rate when mispaired opposite C located next to four G.C base pairs than when correctly paired opposite T next to four A.T base pairs, suggesting that exonuclease mismatch removal specificities may be enhanced to a much greater extent by instabilities of local primer termini than by specific recognition of incorrect base pairs. In polymerase-initiated reactions, biphasic reaction kinetics were observed for the excision of AP within most but not all sequence contexts. Rates of the rapid phases (30-40 s-1) were relatively insensitive to sequence context. Rapid-phase rates reflect the rate constants for exonucleolytic excision of dAPMP from melted primer termini for both correct and incorrect base pairs and were roughly comparable to rates of removal of dAPMP from single-stranded DNA (65-80 s-1). Rates of the slow phases (3-13 s-1) were dependent on sequence context; the slow phase may reflect the rate of switching from the polymerase to the exonuclease active site, or perhaps the conversion of a primer/template terminus from an annealed to a melted state in the exonuclease active site. These data, using wild-type T4 DNA polymerase and two exonuclease-deficient T4 polymerases, support a model in which exonuclease excision occurs on melted primer 3'-termini for both mismatched and correctly matched primer termini, and where specificity favoring removal of terminally mismatched base pairs is determined by the much larger fraction of melted-out primer 3'-termini for mispairs compared to that for correct pairs.
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PMID:Pre-steady-state kinetic analysis of sequence-dependent nucleotide excision by the 3'-exonuclease activity of bacteriophage T4 DNA polymerase. 801 23

The effects of nearest neighbor interactions between a nucleotide base at the primer 3'-terminus and an incoming deoxyribonucleoside triphosphate on DNA polymerase catalyzed insertion were examined. Kinetics of inserting the fluorescent nucleotide analog 2-aminopurine deoxyribonucleotide (dAPMP) and dAMP opposite a template T by 3'-->5' exonuclease-deficient mutants of Klenow fragment (KF-) were measured on primer/templates of identical sequence except for the base pair at the 3'-primer terminus. In addition to its fluorescence properties, 2-aminopurine (AP) is an attractive probe because it is misinserted opposite T by polymerases at much higher frequencies than natural nucleotides. Misinsertion frequencies for AP are on the same order of magnitude as variations in misinsertion frequencies due to changes in local DNA sequence, which makes the statistical significance of these variations easier to document. We have established that changes in the fluorescence of AP can be used to follow the insertion of dAPMP on both steady-state and pre-steady-state time scales. Rates of insertion of dAPMP measured by fluorescence and by a polyacrylamide gel assay were similar and are sensitive to the identity of the base at the 3'-primer twice as fast as insertion following a primer terminus T. The difference in rates arises primarily from differences in kcat values, which were fastest next to G and slowest next to T, while apparent Km values were similar next to each of the 4 different nearest neighbors. The gel assay was used to measure AP misinsertion efficiencies by two methods: (1) by having dAPTP and dATP directly compete for insertion opposite T in the same reaction and (2) by measuring Vmax/Km values for each substrate in separate reactions. The results from the direct competition and separate kinetics measurements are similar. The misinsertion efficiency of dAPMP relative to dAMP opposite a template T was significantly higher next to a 3'-primer terminus G (f(ins) = 0.31 +/- 0.06) than next to T (f(ins) = 0.15 +/- 0.03) for the KF- single mutant (D42A). The corresponding misinsertion efficiencies next to a 3'-primer terminus G and T were 0.20 +/- 0.02 and 0.16, respectively, for the KF- double mutant (D355A, E357A). Relative rates of insertion of dAPMP and dAMP correlate with melting temperatures calculated for nearest neighbor doublets which reflect the relative base-stacking energies. In addition to changes in insertion kinetics, polymerase-DNA dissociation rates varied with the identity of the 3'-primer terminus, differing by as much as 7-20-fold depending on the polymerase and the primer/template.
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PMID:Influence of 5'-nearest neighbors on the insertion kinetics of the fluorescent nucleotide analog 2-aminopurine by Klenow fragment. 821 90

The fluorescence of 2-aminopurine deoxynucleotide positioned in a 3'-terminal mismatch was used to evaluate the pre-steady state kinetics of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase on defined DNA substrates. DNA substrates with one, two, or three preformed terminal mispairs simulated increasing degrees of strand separation at a primer terminus. The effects of base pair stability and local DNA sequence on excision rates were investigated by using DNA substrates that were either relatively G + C- or A + T-rich. The importance of strand separation as a prerequisite to the hydrolysis of a terminal nucleotide was demonstrated by using a unique mutant DNA polymerase that could degrade single-stranded but not double-stranded DNA, unless two or more 3'-terminal nucleotides were unpaired. Our results led us to conclude that the reduced exonuclease activity of this mutant DNA polymerase on duplex DNA substrates is due to a defect in melting the primer terminus in preparation for the excision reaction. The mutated amino acid (serine substitution for glycine at codon 255) resides in a critical loop structure determined from a crystallographic study of an amino-terminal fragment of T4 DNA polymerase. These results suggest an active role for amino acid residues in the exonuclease domain of the T4 DNA polymerase in the strand separation step.
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PMID:Using 2-aminopurine fluorescence and mutational analysis to demonstrate an active role of bacteriophage T4 DNA polymerase in strand separation required for 3' --> 5'-exonuclease activity. 891 May 38

Stopped-flow fluorescence assay was applied to identify conformational changes in the catalytic cycle of DNA polymerase beta (Pol beta), using a synthetic DNA primer/template containing 2-aminopurine (2-AP) at the template position opposite the incoming dNTP. Two phases of fluorescence change were observed in the stopped-flow fluorescence assay of the incorporation of the correct nucleotide dTTP. The rate of the slow phase corresponds to that of product formation. This slow phase was identified as the result of a rate-limiting conformational change step before chemistry because this slow phase was also observed with a dideoxynucleotide at the 3' end of the primer which prevents chemical bond formation. The fast phase was also attributed to a conformational change step since its dependence on [dTTP] is hyperbolic. The rates of the two phases and their dependence on [dTTP] and [Mg2+] suggest that the fast conformational change is induced by the binding of MgdNTP and the slow conformational change is induced by the binding of the catalytic Mg2+ ion. The same biphasic kinetics with different rates were also observed with the thio analog dTTPalphaS and incorrect nucleotides dATP, dGTP, and dCTP. The structural nature for the two conformational changes has been discussed in relation to the available structural information of this enzyme. The results could help to explain how a polymerase controls and achieves its fidelity with a multiple conformational change mechanism.
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PMID:DNA polymerase beta: multiple conformational changes in the mechanism of catalysis. 930 82

DNA polymerases achieve accurate DNA replication through a delicate balance between primer elongation and proofreading. While insufficient proofreading results in DNA replication errors, indiscriminate removal of correct along with incorrect nucleotides is wasteful and may prevent completion of DNA synthesis. The transition between polymerization and proofreading modes is proposed to be governed by a kinetic barrier that prevents proofreading unless the rate of primer elongation is significantly reduced by the presence of an incorrect base pair at the primer-terminus. We have used mutational analysis, coupled with a sensitive, fluorescence-based assay to characterize intermediate steps in the proofreading pathway. A highly fluorescent complex forms between the bacteriophage T4 DNA polymerase and DNA primer-templates labeled at the 3' terminus with the base analog 2-aminopurine. Formation of the fluorescent complex appears to be a rate-determining step in the proofreading pathway and is impaired for several mutator T4 DNA polymerases with amino acid substitutions in the exonuclease domain. Although these mutant DNA polymerases are proficient in hydrolysis, their reduced ability to form the fluorescent complex imposes a higher kinetic barrier. As a consequence, the mutant DNA polymerases proofread less frequently, resulting in more DNA replication errors.
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PMID:Identification of a transient excision intermediate at the crossroads between DNA polymerase extension and proofreading pathways. 952 Mar 96

8-Oxo-7,8-dihydroguanine (8-oxoGua) can base pair with either cytosine (C) or adenine (A) when replicated by DNA polymerases. The 8-oxoGua.A mismatch is extended in preference to the 8-oxoGua.C pair. Using a model 25-mer/36-mer DNA duplex containing either guanine (Gua).C, 8-oxoGua.C, or 8-oxoGua.A base pairs at the primer terminus and A at the standing start position, we found that the pre-steady-state addition of dTTP opposite A following all three base pairs by bacteriophage T7 DNA polymerase exo- showed burst kinetics, suggesting that extension of all three base pairs is controlled by the rate of a step at or before phosphodiester bond formation. Substitution of dTTP alpha S for dTTP yielded modest thio effects of 1-6, suggesting that extension of all three pairs is limited by the rate of the conformational change prior to phosphodiester bond formation. Pre-steady-state values for kpol (maximum polymerization rate) were 120, 12, and 28 s-1, and Kd values were 2, 75, and 22 microM for insertion of dTTP following Gua.C, 8-oxoGua.C, and 8-oxoGua.A base pairs, respectively. Additional analysis of extension was provided by substitution of A in the standing start position by 2-aminopurine (2-AP), a fluorescent base analogue. Comparison of rapid-quench gel-based assays with stopped-flow fluorescence quenching assays suggested that during addition of dTTP opposite 2-AP phosphodiester bond formation was rate-limiting when 8-oxoGua.C or 8-oxoGua.A were the preceding base pairs, while conformational change was rate-limiting when Gua.C was the preceding base pair. Furthermore, the difference in apparent conformational change rates for addition of dTTP opposite 2-AP following the 8-oxoGua base pairs was greater than the differences in their phosphodiester bond formation rates, suggesting that discrimination in extension may be influenced more by conformational change rates than the rates of phosphodiester bond formation in this mispaired system.
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PMID:Pre-steady-state kinetics of nucleotide insertion following 8-oxo-7,8-dihydroguanine base pair mismatches by bacteriophage T7 DNA polymerase exo-. 952 78


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