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)

We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5' to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3' hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.
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PMID:Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates. 1776 50

For DNA polymerases to proofread a misincorporated nucleotide, the terminal 3-4 nucleotides of the primer strand must be separated from the template strand before being bound in the exonuclease active center. Genetic and biochemical studies of the bacteriophage T4 DNA polymerase revealed that a prominent beta-hairpin structure in the exonuclease domain is needed to efficiently form the strand-separated exonuclease complexes. We present here further mutational analysis of the loop region of the T4 DNA polymerase beta-hairpin structure, which provides additional evidence that residues in the loop, namely, Y254 and G255, are important for DNA replication fidelity. The mechanism of strand separation was probed in in vitro reactions using the fluorescence of the base analogue 2-aminopurine (2AP) and mutant RB69 DNA polymerases that have modifications to the beta hairpin, to the exonuclease active site, or to both. We propose from these studies that the beta hairpin in the exonuclease domain of the T4 and RB69 DNA polymerases functions to facilitate strand separation, but residues in the exonuclease active center are required to capture the 3' end of the primer strand following strand separation.
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PMID:Use of 2-aminopurine fluorescence to study the role of the beta hairpin in the proofreading pathway catalyzed by the phage T4 and RB69 DNA polymerases. 1848 71

Rapid chemical quench assays, as well as equilibrium and stopped-flow fluorescence experiments, were performed with an RB69 DNA polymerase (RB69 pol)-primer-template (P/T) complex containing 2-aminopurine (dAP) and a metal exchange-inert Rh(III) derivative of a deoxynucleoside triphosphate (Rh.dTTP). The objective was to determine the effect of catalytic metal ion (A site) occupancy on the affinity of an incoming Rh.dTTP for the RB69 pol-P/T binary complex and on the rate of the conformational change induced by Rh.dTTP binding. With Ca(2+) in the A site, the affinity of the incoming Rh.dTTP for the RB69 pol-P/T binary complex and the conformational change rate can be determined in the absence of chemistry. When Mg(2+) was added to a ternary complex containing Rh.dTTP opposite dAP, the templating base, nucleotidyl transfer occurred, but the rate of product formation was only one-tenth of that found with Mg.dTTP, as determined by rapid chemical quench assays. Rates of conformational change subsequent to formation of a ternary complex, in the absence of chemistry, were estimated from the rate of change in dAP fluorescence with an increase in the Rh.dTTP concentration. We have shown that there is an initial rapid quenching of dAP fluorescence followed by a second phase of dAP quenching, which has nearly the same rate as that of dTMP incorporation, as estimated from rapid chemical quench experiments. We have also demonstrated that the affinity of Rh.dTTP for occupancy of the B metal ion site is dependent on the presence of Ca(2+). However, a saturating Rh.dTTP concentration in the absence of Ca(2+) results in full quenching of dAP fluorescence, whereas a saturating Ca(2+) concentration in the absence of Rh.dTTP gives only partial quenching of dAP fluorescence. The implications of these results for the mechanism of Fingers closing, metal ion binding, and base selectivity are discussed.
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PMID:Effect of A and B metal ion site occupancy on conformational changes in an RB69 DNA polymerase ternary complex. 1922 37

A complete understanding of the kinetic mechanism of fidelity requires comparison of correct and incorrect dNTP incorporation pathways in both the forward and reverse directions. The studies presented here focus on the dNTP-induced conformational step, which has historically been proposed by many to be the major determinant of fidelity. As it was recently highlighted [Tsai, Y. C., and Johnson, K. A. (2006) Biochemistry 45, 9675-9687], chemistry can be the slowest step in the forward direction of the correct dNTP incorporation pathway, yet the corresponding microscopic rate constant would not contribute toward fidelity in the case when the reverse rate of the conformational step is slower than chemistry. Here we use a stopped-flow technique to directly measure the reverse rate of the conformational step in the DNA polymerase beta (Pol beta) kinetic pathway. Extensive pre-steady-state kinetic studies presented include the utilization of 2-aminopurine-labeled DNA substrates, 2-aminopurine nucleotide triphosphate, a nonhydrolyzable nucleotide analogue dAMPCPP, and a rapid sequential mixing reaction scheme. Additionally, the effect of mismatched dNTPs, various metal ions, and the presence of the 3'-terminal hydroxyl group of the primer on the rate of the reverse "opening" conformational step were analyzed. Our analyses indicate that reverse "opening" is drastically facilitated in the presence of mismatched ternary complexes, which is in agreement with the hypothesis that the ternary complex is destabilized by the presence of incorrect dNTP. By analysis of the relative magnitudes of chemistry and reverse "opening" in the presence of both matched and mismatched matched ternary complexes, this work further validates that, for Pol beta, fidelity is dictated by the differences in free energy required to reach the highest energy transition state of the chemical step.
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PMID:Contribution of the reverse rate of the conformational step to polymerase beta fidelity. 1923 36

DNA polymerase (pol) iota, a member of the mammalian Y-family of DNA polymerases involved in translesion DNA synthesis, has been previously suggested to peculiarly utilize Hoogsteen base pairing for DNA synthesis opposite template purines, unlike pols eta and kappa, which utilize Watson-Crick (W-C) base pairing. To investigate the possible roles of Hoogsteen, W-C, and wobble base-pairing modes in the selection of nucleotides opposite template pyrimidines by human pol iota, we carried out kinetic analyses of incorporation of modified purine nucleoside triphosphates including 7-deazapurines, inosine, 2-aminopurine, 2,6-diaminopurine, and 6-chloropurine, which affect H-bonding in base-pair formation opposite template pyrimidines. Carbon substitution at the N7 atom of purine nucleoside triphosphates, which disrupts Hoogsteen base pairing, only slightly inhibited DNA synthesis opposite template pyrimidines by pol iota, which was not substantially different from human pols eta and kappa. Opposite template T, only the relative wobble stabilities (inferred from the potential numbers of H-bonding, steric, and electrostatic interactions but not measured) of base pairs were positively correlated to the relative efficiencies of nucleotide incorporation by pol iota but not the relative W-C or Hoogsteen stabilities, unlike pols eta and kappa. In contrast, opposite C, only the relative W-C stabilities of base pairs were positively correlated to the relative efficiencies of nucleotide incorporation by pol iota, as with pols eta and kappa. These results suggest that pol iota might not indispensably require Hoogsteen base pairing for DNA synthesis opposite pyrimidines but rather might prefer wobble base pairing in the selection of nucleotides opposite T and W-C base pairing opposite C.
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PMID:Kinetic analysis of base-pairing preference for nucleotide incorporation opposite template pyrimidines by human DNA polymerase iota. 1937 29

In addition to their capacity for template-directed 5' --> 3' DNA synthesis at the polymerase (pol) site, DNA polymerases have a separate 3' --> 5' exonuclease (exo) editing activity that is involved in assuring the fidelity of DNA replication. Upon misincorporation of an incorrect nucleotide residue, the 3' terminus of the primer strand at the primer-template (P/T) junction is preferentially transferred to the exo site, where the faulty residue is excised, allowing the shortened primer to rebind to the template strand at the pol site and incorporate the correct dNTP. Here we describe the conformational changes that occur in the primer strand as it shuttles between the pol and exo sites of replication-competent Klenow and Klentaq DNA polymerase complexes in solution and use these conformational changes to measure the equilibrium distribution of the primer between these sites for P/T DNA constructs carrying both matched and mismatched primer termini. To this end, we have measured the fluorescence and circular dichroism spectra at wavelengths of >300 nm for conformational probes comprising pairs of 2-aminopurine bases site-specifically replacing adenine bases at various positions in the primer strand of P/T DNA constructs bound to DNA polymerases. Control experiments that compare primer conformations with available x-ray structures confirm the validity of this approach. These distributions and the conformational changes in the P/T DNA that occur during template-directed DNA synthesis in solution illuminate some of the mechanisms used by DNA polymerases to assure the fidelity of DNA synthesis.
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PMID:Local conformations and competitive binding affinities of single- and double-stranded primer-template DNA at the polymerization and editing active sites of DNA polymerases. 1941 Dec 53

The fluorescence of the base analog 2-aminopurine (2AP) is used in highly sensitive assays to determine kinetic parameters for DNA polymerase catalyzed reactions, including exonucleolytic proofreading and nucleotide binding and incorporation. Since 2AP fluorescence can also be used to probe DNA polymerase-induced conformational changes in 2AP-labeled DNA substrates, reaction steps that occur before product formation can be detected. Instruction is provided here in the use of 2AP fluorescence in steady-state and presteady-state assays to study DNA polymerase function and DNA replication.
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PMID:The use of 2-aminopurine fluorescence to study DNA polymerase function. 1956 18

Escherichia coli DinB (DNA polymerase IV) possesses an enzyme architecture resulting in specialized lesion bypass function and the potential for creating -1 frameshifts in homopolymeric nucleotide runs. We have previously shown that the mutagenic potential of DinB is regulated by the DNA damage response protein UmuD(2). In the current study, we employ a pre-steady-state fluorescence approach to gain a mechanistic understanding of DinB regulation by UmuD(2). Our results suggest that DinB, like its mammalian and archaeal orthologs, uses a template slippage mechanism to create single base deletions on homopolymeric runs. With 2-aminopurine as a fluorescent reporter in the DNA substrate, the template slippage reaction results in a prechemistry fluorescence change that is inhibited by UmuD(2). We propose a model in which DNA templates containing homopolymeric nucleotide runs, when bound to DinB, are in an equilibrium between non-slipped and slipped conformations. UmuD(2), when bound to DinB, displaces the equilibrium in favor of the non-slipped conformation, thereby preventing frameshifting and potentially enhancing DinB activity on non-slipped substrates.
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PMID:UmuD(2) inhibits a non-covalent step during DinB-mediated template slippage on homopolymeric nucleotide runs. 2046 52

DNA polymerases catalyze the incorporation of deoxynucleoside triphosphates into a growing DNA chain using a pair of Mg(2+) ions, coordinated at the active site by two invariant aspartates, whose removal by mutation typically reduces the polymerase activity to barely detectable levels. Using two stopped-flow fluorescence assays that we developed previously, we have investigated the role of the carboxylate ligands, Asp(705) and Asp(882), of DNA polymerase I (Klenow fragment) in the early prechemistry steps that prepare the active site for catalysis. We find that neither carboxylate is required for an early conformational transition, reported by a 2-aminopurine probe, that takes place in the open ternary complex after binding of the complementary dNTP. However, the subsequent fingers-closing step requires Asp(882); this step converts the open ternary complex into the closed conformation, creating the active-site geometry required for catalysis. Crystal structures indicate that the Asp(882) position changes very little during fingers-closing; this side chain may therefore serve as an anchor point to receive the dNTP-associated metal ion as the nucleotide is delivered into the active site. The Asp(705) carboxylate is not required until after the fingers-closing step, and we suggest that its role is to facilitate the entry of the second Mg(2+) into the active site. The two early prechemistry steps that we have studied take place normally at very low Mg(2+) concentrations, although higher concentrations are needed for covalent nucleotide addition, consistent with the second metal ion entering the ternary complex after fingers-closing.
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PMID:Distinct roles of the active-site Mg2+ ligands, Asp882 and Asp705, of DNA polymerase I (Klenow fragment) during the prechemistry conformational transitions. 2108 97

The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased "breathing" at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.
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PMID:Structure of the 2-aminopurine-cytosine base pair formed in the polymerase active site of the RB69 Y567A-DNA polymerase. 2202 3


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