FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1588264401> ?p ?o ?g. }

• W1588264401 endingPage "31243" @default.
• W1588264401 startingPage "31235" @default.
• W1588264401 abstract "Amino acids Tyr254 and Tyr390 of φ29 DNA polymerase belong to one of the most conserved regions in eukaryotic-type DNA polymerases. In this paper we report a mutational study of these two residues to address their role in nucleotide selection. This study was carried out by means of a new kinetic analysis that takes advantage of the competition between DNA polymerization and 3′→5' exonuclease activity to measure the Km values for correct and incorrect nucleotides in steady-state conditions. This method is valid for any 3′→5' exonuclease-containing DNA polymerase, without any restriction concerning catalytic rates of nucleotide incorporation.The results showed that the discrimination factor achieved by φ29 DNA polymerase in the nucleotide binding step of DNA polymerization is 2.4 × 103, that is, a wrong nucleotide is bound with a 2.4 × 103-fold lower affinity than the correct one. Mutants Y254F, Y390F, and Y390S showed discrimination values of 7.0 × 102, >1.9 × 103, and 2.9 × 102, respectively. The reduced accuracy of nucleotide binding produced by mutations Y254F and Y390S lead us to propose that φ29 DNA polymerase residues Tyr254 and Tyr390, highly conserved in eukaryotic-type DNA polymerases, are involved in nucleotide binding selection, thus playing a crucial role in the fidelity of DNA replication. Comparison of the discrimination factors of mutants Y390S and Y390F strongly suggests that the phenyl ring of Tyr390 is directly involved in checking base-pairing correctness of the incoming nucleotide. Amino acids Tyr254 and Tyr390 of φ29 DNA polymerase belong to one of the most conserved regions in eukaryotic-type DNA polymerases. In this paper we report a mutational study of these two residues to address their role in nucleotide selection. This study was carried out by means of a new kinetic analysis that takes advantage of the competition between DNA polymerization and 3′→5' exonuclease activity to measure the Km values for correct and incorrect nucleotides in steady-state conditions. This method is valid for any 3′→5' exonuclease-containing DNA polymerase, without any restriction concerning catalytic rates of nucleotide incorporation. The results showed that the discrimination factor achieved by φ29 DNA polymerase in the nucleotide binding step of DNA polymerization is 2.4 × 103, that is, a wrong nucleotide is bound with a 2.4 × 103-fold lower affinity than the correct one. Mutants Y254F, Y390F, and Y390S showed discrimination values of 7.0 × 102, >1.9 × 103, and 2.9 × 102, respectively. The reduced accuracy of nucleotide binding produced by mutations Y254F and Y390S lead us to propose that φ29 DNA polymerase residues Tyr254 and Tyr390, highly conserved in eukaryotic-type DNA polymerases, are involved in nucleotide binding selection, thus playing a crucial role in the fidelity of DNA replication. Comparison of the discrimination factors of mutants Y390S and Y390F strongly suggests that the phenyl ring of Tyr390 is directly involved in checking base-pairing correctness of the incoming nucleotide. One of the most remarkable properties of DNA polymerases is their ability to replicate DNA with a very high fidelity (10-5-10-8 errors/nucleotide) (Loeb and Kunkel, 1982; Echols and Goodman, 1991). This is much less than the error frequency of 2 × 10-1-6 × 10-3 errors per nucleotide polymerized, predicted on the basis of energy differences between correct and incorrect base pairs (Loeb and Kunkel, 1982). The accuracy of DNA polymerization is based upon template instruction, that is changed after each nucleotide incorporation event. Taking into account that DNA polymerases synthesize DNA at high velocity (100-1000 nucleotides/s) (Kornberg and Baker, 1992), their substrate specificity has to change very rapidly, while maintaining a strong discrimination against wrong nucleotides. The molecular mechanisms underlying this amazing behavior are poorly understood. The considerable accuracy of DNA synthesis is achieved by the sequential operation of two fidelity mechanisms: (a) selection of the correct deoxynucleoside triphosphate in the polymerization reaction (base selection) and (b) exonucleolytic removal of an incorrectly inserted deoxynucleoside monophosphate from the end of the growing chain (editing). For base selection, the most likely discrimination principle is a strict demand for the precise geometry of the Watson-Crick base pair. This requirement might be manifested either in a more rapid dissociation from the enzyme active site of an incorrect nucleotide or in a considerably slower phosphodiester bond formation for mispaired bases, or most likely both. Additional mechanisms based on intermediate conformational changes have been described to discriminate against incorrect incorporation (Wong et al., 1991). Besides, the addition of the next correct nucleotide onto a mismatch occurs at a very slow rate, stalling the polymerase after misincorporation, and, therefore, allowing time for the exonuclease activity to attack the mispaired primer terminus (Donlin et al., 1991; Carroll et al., 1991; reviewed in Johnson(1993)). In the absence of structural data, site-directed mutagenesis is a powerful tool to elucidate the contribution of individual amino acid residues of the DNA polymerization site to the fine nucleotide selection exhibited by most replication enzymes. However, very few studies have addressed properly the fidelity of mutant DNA polymerases. The high catalytic rate of nucleotide incorporation compromises the utilization of standard steady-state methods to describe the kinetic properties of DNA polymerization. Besides this, most DNA polymerases contain a 3′→5' exonuclease activity that competes with DNA synthesis, this competition being unbalanced toward exonucleolytic degradation in the case of polymerization mutants, where nucleotide incorporation is impaired. Whereas this second point can be overcome by using exonuclease-deficient mutants, the study of very fast reactions in appropriate time windows seems to require rapid quench-flow techniques (Patel et al., 1991; Wong et al., 1991; Eger et al., 1991; Hsieh et al., 1993). In this paper we report that meaningful biochemical information can be obtained from steady-state kinetics when the exonuclease activity is allowed to compete with DNA polymerization. We are using this approach to study the molecular mechanisms of DNA replication fidelity using φ29 DNA polymerase as model. This polymerase provides a simple and well-characterized model for studying template-directed DNA synthesis and identifying which amino acid side chains participate in substrate selection and catalysis of phosphodiester bond formation. Extensive site-directed mutagenesis studies based on amino acid sequence comparison have allowed us to define the functional role for most of the residues belonging to highly conserved motifs in DNA polymerases (reviewed in Blanco and Salas(1995)). This large collection of φ29 DNA polymerase mutants is now available to elucidate the contribution of individual amino acids of the active center to the mechanisms maintaining fidelity. Furthermore, a basic kinetic and fidelity description of both DNA replication and protein-primed initiation has been reported for φ29 DNA polymerase (Esteban et al., 1993). Recent structural and kinetic data indicate that polymerases achieve selectivity by mechanisms that differ in detail, although, from the available evidence, it is accepted that all polymerases follow general fidelity principles for base selection and editing (Echols and Goodman, 1991). Therefore, the conclusions to be drawn from φ29 DNA polymerase (a prototype enzyme for eukaryotic-type DNA polymerases) will shed some light into the basic mechanisms maintaining fidelity. For the present study we have chosen two φ29 DNA polymerase residues: Tyr254 and Tyr390. They belong to the highly conserved motifs DXXSLYP (region 1 according to Blanco et al.(1991), region A according to Delarue et al.(1990)) and KXXXNSLYG (region 2a according to Blanco et al.(1991), region B according to Delarue et al.(1990)), respectively. The conservative mutations Y254F, Y390S, and Y390F were reported to show a defective dNTP binding affinity, closely related to a template-dependent hypersensitivity to nucleotide analogs, such as 2-(p-n-butylanilino)dATP and N2-(p-n-butylphenyl)dGTP (Blasco et al., 1992). Therefore, these DNA polymerase mutants appeared as ideal candidates for fidelity studies. Ultrapure unlabeled dNTPs were from Pharmacia Biotech Inc.; [γ-32P]ATP (3,000 Ci/mmol) was obtained from Amersham International Plc. T4 polynucleotide kinase was purchased from New England Biolabs. Wild-type φ29 DNA polymerase and mutant derivatives Y254F, Y390F, and Y390S (Blasco et al., 1992) were purified as described by Lázaro et al.(1995). Complementary single-stranded oligonucleotides SP1 (15-mer) (5′-GATCACAGTGAGTAC) and SP1c+6 (21-mer) (5′-TCTATTGTACTCACTG TGATC) were synthesized and purified by 20% polyacrylamide gel electrophoresis. Oligonucleotide SP1 was 5′-labeled with [γ-32P]ATP and T4 polynucleotide kinase and further purified by polyacrylamide gel electrophoresis. The specific activity obtained was 2.5 × 105 cpm/pmol. The primer-template 5′-32P-labeled SP1/SP1c+6 molecule was obtained by hybridization between SP1 and SP1c+6. φ29 DNA polymerases, wild-type or mutants, were allowed to interact with template-primer DNA (SP1/SP1c+6) at equilibrium by preincubating for 5 min at 0°C all the components of the final reaction mixture, except the metal activator. Then the incubation mixture was tempered to 30°C, and the reactions started by adding MgCl2. The reaction samples contained, in 12 μl, 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml bovine serum albumin, 1 nM 5′-32P-labeled SP1/SP1c+6, 75 nM wild-type or mutant φ29 DNA polymerase, 10 mM MgCl2, 20 nM dATP, and 10 μM dCTP, to prevent exonucleolytic degradation of the primer DNA. In the case of wild-type and mutant Y390S proteins, the incubation times ranged from 1 to 5 min and from 2 s to 4 min, in the case of mutants Y254F and Y390F. Samples were stopped by adding EDTA up to 100 mM and analyzed by 8 M urea-20% polyacrylamide gel electrophoresis, autoradiography, and densitometry. DNA template-primer and φ29 DNA polymerase were preincubated as described above. Reaction mixtures contained, in 12 μl, 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml bovine serum albumin, 75 nM φ29 DNA polymerase, 10 mM MgCl2, 2 μM dATP, and 10 μM dCTP, and a template-primer concentration ranging from 1 to 100 nM. After tempering for 30 s at 30°C, reactions were started by adding the metal activator and stopped after 2 min by adding EDTA up to 100 mM. The samples were processed as described above. φ29 DNA polymerase and the DNA template-primer were preincubated as described above. Then, the incubation mixture was tempered to 30°C, and the reactions started by adding MgCl2. The final reaction mixture contained, in 12 μl, 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml bovine serum albumin, 1 nM 5′-32P-labeled SP1/SP1c+6, 75 nM wild-type or mutant φ29 DNA polymerase, 10 mM MgCl2, and various concentrations of dATP, ranging at least two orders of magnitude. In every experiment, 10 μM dCTP was included to prevent exonucleolytic degradation of the DNA template-primer. After incubation at 30°C for 15 s in the case of φ29 DNA polymerase mutants Y390F and Y254F, or 2 min in the case of wild-type or mutant Y390S, reactions were stopped with EDTA up to 100 mM. The samples were analyzed by 8 M urea-20% polyacrylamide gel electrophoresis and autoradiography. The amount of the different elongation products as well as the nonelongated primer was quantified by densitometry of the autoradiograph. The ratio between the 17-mer and the 16-mer elongation products (two-nucleotide and one-nucleotide additions on the SP1 primer, respectively) was calculated and plotted against the dATP concentration, according to the theoretical method described in the text. The graphs were fitted to rectangular hyperbolas by nonlinear regression analysis using both the Newton and the steepest-descent methods. The kinetic parameters were obtained as described in the text. The reaction mixtures were as described above but, in this case, 20 μM dATP was included. This concentration was proven to be enough for the two first correct incorporations. For the incorrect nucleotide insertion on the third position of the template, different dCTP concentrations were assayed within a range of at least one order of magnitude. The dATP concentration present in the reaction mixtures also allows the incorporation of the next correct nucleotide onto the mismatch. The ratio between the error-containing elongation products (misinsertion, 18-mer, and mismatch elongation, 19-mer) and the last correctly paired elongation product (17-mer) was calculated and plotted against dCTP (incorrect nucleotide) concentration. The experimental data were fitted to a rectangular hyperbola to calculate the kinetic parameters described in the text. In the case of mutant Y390F, the highest dCTP concentrations assayed produced a severe inhibition of nucleotide insertion. Therefore, these concentrations were discarded to calculate nucleotide insertion parameters and only the initial slope of the curve was considered. Kinetics of single nucleotide incorporation catalyzed by DNA polymerases follow single or double exponential behavior (depending on experimental conditions), that usually reach equilibrium in the millisecond time scale (Patel et al., 1991). This rapid nucleotide insertion makes the steady-state studies difficult to interpret, because, in most of the cases, the rate-limiting step is DNA dissociation from the enzyme. Therefore, the information concerning the fast catalytic step is usually missed or mistaken. We have studied the influence of 3′→5' exonuclease activity on DNA polymerization kinetics. The mathematical treatment of pre-steady-state kinetics becomes considerably more complex when the exonuclease activity is included. However, we will show that, after the transient pre-steady-state, the final steady-state that is reached offers unambiguous information of some kinetic parameters governing DNA polymerization. This model represents a steady-state analysis of the competition between DNA polymerization and exonuclease activity (see below). Therefore, in order for this model to be applicable, two main conditions have to be fulfilled: DNA polymerase-DNA complex dissociation must not be rate-limiting, and solely the final steady-state phase of the time course has to be considered. First, we will describe the validity of this approach to study the kinetic parameters of correct and incorrect nucleotide incorporation catalyzed by the wild-type φ29 DNA polymerase. Then, we will apply this model to study the fidelity of Y254F, Y390S, and Y390F mutant derivatives of φ29 DNA polymerase, previously described as deficient in nucleotide binding. We have considered the insertion of a correct nucleotide on a homopolymer tract as template, in the presence of the exonuclease activity. Two conditions should be imposed. First, the initial primer (D0) has to be stable against exonucleolytic degradation, in order to keep constant the total amount of DNA template-primer in the reaction mixture. Second, the actual template has to be longer than the homopolymeric tract, to stop polymerization before reaching the end of the template. The reaction pathway that describes this system is shown in Fig. SI. In this scheme, E•Di stands for the binary complex formed by the i-mer primer DNA and DNA polymerase; kcat and kexa stand for the catalytic rates of DNA polymerization and exonuclease reactions, respectively; k1 and k-1 represent the association and dissociation rates, respectively, of nucleotide binding; n is the number of consecutive nucleotide incorporations allowed on the homopolymeric tract. Using steady-state kinetic analysis, the concentration of the different species can be derived as: Di=DT(kexokcat)n-i⋅(KMN)n-i1+(KMN+1)⋅∑i=0n-1(kexokcat)n-i⋅(KMN)n-i-1(Eq. 1) (Di⋅N)=DT(kexokcat)n-i⋅(KMN)n-i-11+(KMN+1)⋅∑i=0n-1(kexokcat)n-i⋅(KMN)n-i-1(Eq. 2) In these formulae, Di and (Di•N) represent the concentration of a certain DNA intermediate, i-bases long, and the complex i-mer DNA primer with the next nucleotide to be incorporated bound in its position, not yet catalyzed (see Fig. SI); both Di and (Di•N) stand for the corresponding complexes with DNA polymerase; Dt is the total amount of DNA polymerase/DNA complexes; N stands for dNTP concentration, and Km = (k-1+ kcat)/k1. These equations predict the steady-state distribution of the different elongated complexes as a function of kcat/kexa, Km, and nucleotide concentration. This theoretical distribution can be compared to experimental ones, generated at different dNTP concentrations, to fit the kinetic parameters kcat/kexa-1 and Km. A more straightforward approach can be followed by considering the ratio between the concentration of the two last elongated complexes (Dn and Dn): DnDn-1+(Dn-1⋅N)=kcatkexo⋅NN+Km(Eq. 3) The relative proportion between the two last DNA species appears as a function of nucleotide concentration, where kcat/kexa-1 and Km can be obtained from a nonlinear least square fit to the rectangular hyperbola. For a practical approach to this model, we used as DNA substrate a 5′-labeled 15/21-mer primer-template oligonucleotide, which allows the consecutive incorporation of two correct dAMP residues; that is, the homopolymeric tract is restricted to two nucleotides (see Fig. SII and Materials and Methods). Primer degradation was prevented by including 10 μM dCTP in all the experiments (this concentration was proven not to interfere with the incorporation of the correct dATP nucleotide; not shown). Using this experimental system, Dn/Dn (see Fig. SI) becomes D2/D1. As described previously, the mathematical treatment presented here is valid only if the steady-state has been reached, and the DNA polymerase/DNA complex dissociation is not rate-limiting in this steady-state. These two points were addressed and confirmed experimentally. The incubation time required to reach the steady-state was determined by following the time course of the reaction. In this experiment, a very low concentration of dATP (20 nM) was added, so as to evaluate the time course at a very slow forward rate, the enzyme being far away from its maximal velocity. The diagnostic feature of the steady-state is the invariance of all the elongation product concentrations. In our case, due to the low dATP concentration, the only elongation product is D1. As shown in Fig. 1, the intensity of the bands corresponding to D1 and D0 is invariant from the first time tested (1 min). To evaluate whether dissociation or dissociation-reassociation are rate-limiting steps in the reaction, another experiment was carried out with increasing amounts of template-primer DNA and a fixed concentration of φ29 DNA polymerase. As shown in Fig. 2, at the highest DNA concentrations, the relative proportions of elongated primers ((D1+ D2)/D0) is very low, indicating that φ29 DNA polymerase is titrated out. However, the proportion between the longest elongation product and the shortest one (D2/D1) is independent from the DNA polymerase/DNA ratio. These results indicate that φ29 DNA polymerase is not being dissociated from the template, and, therefore, complex dissociation does not influence the final steady-state. Correct nucleotide insertion kinetics were carried out at different dATP concentrations, which bracketed the Km value for this nucleotide, allowing the system to reach steady-state conditions. The ratio between the intensity of bands D2 and D1 was calculated and plotted against dATP concentration (see Fig. 3). Experimental data were fitted to by nonlinear regression analysis, and Km and kcat/kexa were estimated as 0.5 μM and 2.6, respectively (see Table 1).Tabled 1 Open table in a new tab To study the incorporation of a wrong nucleotide, the same 5′-labeled 15/21-mer primer-template molecule was used. In this case, a sufficient concentration (20 μM) of dATP was included to allow the incorporation of the first two correct nucleotides. The misincorporation of C opposite A in the template was monitored at different dCTP concentrations. The dATP concentration present in the experiment allows the elongation of the mispairs using the next T in the template (see Fig. SIII). In this model, kerr and kext are the catalytic rates for inserting an incorrect nucleotide and extending it with a correct one, respectively; k2 and k-2 stand for the association and dissociation rates, respectively, of dCTP binding; k3 and k-3 represent the association and dissociation rates, respectively, for binding the next correct nucleotide; kexa, kexol, and kexo2err are the catalytic rates of the exonuclease reaction on the properly paired primer terminus, on the mismatched one, and on the correctly elongated mismatch, respectively. The steady-state analysis of this system leads to the following equations: D2=DT⋅kcat2⋅kexol⋅kexo2⋅N2⋅Kmerr⋅KmextS(Eq. 4) (D2⋅I)=DT⋅kcat2⋅kexol⋅kexo2⋅N2⋅I⋅KmextS(Eq. 5) D3=DT⋅kcat2⋅kerr⋅kexo2⋅N2⋅I⋅KmextS(Eq. 6) (D3.N)=DT⋅kcat2⋅kerr⋅kexo2⋅N3⋅IS(Eq. 7) D4=DT⋅kcat2⋅kerr⋅kext⋅N3⋅IS(Eq. 8) where D2, (D2•I), D3, (D3•N), and D4 are the different elongation products in the polymerization process (positions 17, 18, and 19, respectively; see oligonucleotide sequence in Fig. SIII). These DNA products are assumed to be always forming a complex with the enzyme. Dt is the total amount of DNA polymerase/DNA complexes. N and I represent the correct and incorrect nucleotide concentrations, respectively. The different affinity constants are defined as follows: Kmerr=k-2+kerrk2(Eq. 9) and Kmerr=k-3+kextk3(Eq. 10) S stands for the following expression: S=kexo2⋅kexol⋅kexo2⋅Kmext⋅Kmerr⋅Km2+kexo2⋅kexol⋅kexo2⋅Kmext⋅Kmerr⋅Km⋅N+kexo⋅kexol⋅kexo2⋅kcat⋅Kmext⋅Kmerr⋅Km⋅N+kerr⋅kexo2⋅kcat2⋅Kmext⋅N2⋅I+kexo⋅kexol⋅kexo2⋅kcat⋅Kmext⋅Kmerr⋅N2+kexol⋅kexo2⋅Kcat2⋅Kmext⋅Kmerr⋅N2+kerr⋅kexo2⋅kcat2⋅N3⋅I+kext⋅kerr⋅kcat2⋅N3⋅I+kexol⋅kexo2+kcat2⋅Kmext⋅N2⋅I(Eq. 11) Km stands for the Michaelis constant of the two first correct nucleotide insertions, as defined for. The ratio between the error-containing products (positions D3 plus D4) and the correct nucleotide insertion (position D2) gives us the following result: D3+(D3⋅N)+D4D2+(D2⋅I)=kerr⋅(kext⋅N+kexo2⋅N+kexo2⋅Kmext)kexol⋅kexo2⋅Kmext⋅IKmerr+I(Eq. 12) Therefore, this ratio versus the concentration of the incorrect nucleotide (I) would give a rectangular hyperbola, from which the Km for the incorrect nucleotide (Km) can be obtained. The incubation times required to reach the steady-state, as well as the absence of a DNA dissociation rate-limiting step, were assessed as described for the correct nucleotide (not shown). The incorporation of the incorrect nucleotide was measured at different dCTP concentrations, and the ratio (D3+ D4)/D2 was calculated. The experimental data were fitted to by nonlinear regression analysis. The results are shown in Fig. 4(see also Table 1). φ29 DNA polymerase mutants Y254F, Y390F, and Y390S have been described as affected in dNTP binding (Blasco et al., 1992). Nevertheless, their deficiency in dNTP affinity could not be precisely assessed because Km could not be determined either for the wild type or for the mutant DNA polymerases. We have applied the exonuclease-polymerization competition model, presented above, to study the steady-state kinetics of correct and incorrect nucleotide incorporation catalyzed by φ29 DNA polymerase mutants Y254F, Y390F, and Y390S. As described for the wild-type enzyme, the absence of a rate-limiting DNA dissociation step and the incubation time required to reach steady-state conditions were evaluated experimentally for each mutant (not shown). In the case of mutants Y254F and Y390F, we observed that long incubation periods led to exonucleolytic degradation of most of the DNA primer substrate (not shown). This is probably due to their reduced DNA polymerization rate, that renders the standard dCTP concentration insufficient for protecting against their exonuclease activity. In order to avoid potential interferences with the incorporation of the correct nucleotide and taking into account that steady-state conditions were achieved after 10 s (not shown), we decided to use shorter incubation times (15 s) rather than to increase dCTP concentrations. It is worthy to note that, using this experimental approach, the incubation time is not relevant, as long as the steady-state has been reached. The incorporation of correct (dATP) and incorrect (dCTP) nucleotides were evaluated at different dNTP concentrations, and the experimental data were analyzed as described for the wild-type enzyme. The results are shown in Fig. 5. Concerning the incorporation of the correct nucleotide, mutants Y254F, Y390F, and Y390S showed 10, 4.6, and 14-fold, respectively, reduction in dNTP affinity, compared to the wild-type φ29 DNA polymerase (see Table 1). The discrimination factor achieved during nucleotide binding can be obtained from the different affinity for correct and incorrect nucleotides and represented by fdis = Km (incorrect)/Km (correct). This factor was calculated for the wild-type and mutant φ29 DNA polymerases and is shown in Table 1. Proteins Y254F and Y390S showed a reduced discrimination ability (7.0 × 102 and 2.9 × 102, respectively), whereas the discrimination value of Y390F (>1.9 × 103) was quite similar to the one of the wild-type enzyme (2.4 × 103). The quantification of these effects is also shown in Table 1, as relative discrimination factors, that were calculated relative to the wild-type enzyme. Since the first reports of in vitro studies concerning the fidelity of DNA polymerization (Trautner et al., 1962; Hall and Lehman, 1968; Brutlag and Kornberg, 1972), a large number of DNA polymerases have been purified and their accuracies measured experimentally (reviewed in Echols and Goodman(1991), Kunkel(1992), Johnson(1993)). Based on these studies, the general strategies for achieving faithful DNA synthesis have been enlightened (reviewed in Johnson(1993)). Presently, many DNA polymerase studies are devoted to understand how the active center of these enzymes promotes such a highly selective reaction mechanism. In this work, we have presented a new model for studying DNA polymerization kinetics in steady-state conditions, and we have applied it to get some insight into the molecular mechanisms of fidelity, operating at the active center of φ29 DNA polymerase. Various steady-state methods to study DNA polymerization on synthetic templates have been used widely (Boosalis et al., 1987; El-Deiry et al., 1988; Polesky et al., 1990; Reha-Krantz et al., 1991; Bakhanashvili and Hizi, 1992a and 1992b; Creighton et al., 1992; Varela-Echavarría et al., 1992; Copeland et al., 1993; Dong et al., 1993a, 1993b; Khan et al., 1994; Astatke et al., 1995). The information that can be obtained from these analyses is often compromised by the fact that DNA polymerase/DNA complex dissociation is rate-limiting in most of the steady-state kinetics of DNA polymerization. On the other hand, the presence of a 3′→5' exonuclease activity associated with many DNA polymerases makes it more difficult to correlate the experimental data with the kinetic parameters governing DNA polymerization. In this situation, an apparent Km is usually reported that simply reflects the nucleotide concentration at which half of the maximal rate of nucleotide incorporation is achieved. However, this parameter has no explicit relation with nucleotide binding affinity. In this paper, we propose a general method to calculate the true Km values for correct and incorrect nucleotides overcoming these limitations. As true Km, we consider its original explicit definition: Km = (k-1+ kcat)/k1. The kinetic analysis proposed here takes advantage of the 3′→5' exonuclease activity to compete DNA polymerization, leading to a true steady-state. Once this state has been reached, the relative amount of each DNA elongation product remains constant with time. This situation should not be taken as an equilibrium state, as there is a continuous (steady) interconversion between the different DNA products by means of deoxynucleoside triphosphate use and monophosphate production. Therefore, in order to apply this method to different systems, it has to be explicitly verified that the early transient stage of DNA product rearrangement is overcome. This requirement was specifically met with the wild-type and mutant φ29 DNA polymerases presented in this study. Another key feature of this model, noticeable from Schemes I, II, and III, is the absence of dissociation rates (koff). As described in the text, all the DNA species are considered as DNA polymerase/DNA complexes, therefore, competent for nucleotide insertion or exonucleolytic degradation. The avoidance of dissociation and association steps between DNA polymerase and DNA allows a more straightforward interpretation of the experimental data in terms of the catalytically relevant rate constants. However, the experimental conditions have to be designed in order to fulfill the requirement for a DNA dissociation-independent reaction pathway. As demonstrated in the DNA titration experiment (this paper), φ29 DNA polymerase is able to remain stably bound to the template/primer, even when it has been stalled by the absence of the next correct nucleotide. In this experiment, the probability of inserting one or two nucleotides was measured at different DNA polymerase/DNA ratios. If the insertion of the second nucleotide is mediated by DNA polymerase dissociation and reasso" @default.
• W1588264401 created "2016-06-24" @default.
• W1588264401 creator A5052448979 @default.
• W1588264401 creator A5056783795 @default.
• W1588264401 creator A5079506679 @default.
• W1588264401 creator A5089344832 @default.
• W1588264401 date "1995-12-01" @default.
• W1588264401 modified "2023-09-26" @default.
• W1588264401 title "A Novel Kinetic Analysis to Calculate Nucleotide Affinity of Proofreading DNA Polymerases:" @default.
• W1588264401 cites W1480442757 @default.
• W1588264401 cites W1481052392 @default.
• W1588264401 cites W1490908705 @default.
• W1588264401 cites W1494432265 @default.
• W1588264401 cites W1499552914 @default.
• W1588264401 cites W1503567487 @default.
• W1588264401 cites W1511239607 @default.
• W1588264401 cites W1541732942 @default.
• W1588264401 cites W1568159422 @default.
• W1588264401 cites W1597138031 @default.
• W1588264401 cites W1967653471 @default.
• W1588264401 cites W1979511146 @default.
• W1588264401 cites W1990083018 @default.
• W1588264401 cites W1994304491 @default.
• W1588264401 cites W1996914885 @default.
• W1588264401 cites W2003469154 @default.
• W1588264401 cites W2008554680 @default.
• W1588264401 cites W2020152372 @default.
• W1588264401 cites W2023917633 @default.
• W1588264401 cites W2025953091 @default.
• W1588264401 cites W2032122437 @default.
• W1588264401 cites W2032647696 @default.
• W1588264401 cites W2050445949 @default.
• W1588264401 cites W2056455068 @default.
• W1588264401 cites W2066630664 @default.
• W1588264401 cites W2071625124 @default.
• W1588264401 cites W2074507088 @default.
• W1588264401 cites W2089898350 @default.
• W1588264401 cites W2094468496 @default.
• W1588264401 cites W2108150290 @default.
• W1588264401 cites W2138476183 @default.
• W1588264401 cites W2157114285 @default.
• W1588264401 cites W2174994538 @default.
• W1588264401 cites W2580627695 @default.
• W1588264401 cites W25978485 @default.
• W1588264401 cites W3141642568 @default.
• W1588264401 cites W4256385571 @default.
• W1588264401 doi "https://doi.org/10.1074/jbc.270.52.31235" @default.
• W1588264401 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8537389" @default.
• W1588264401 hasPublicationYear "1995" @default.
• W1588264401 type Work @default.
• W1588264401 sameAs 1588264401 @default.
• W1588264401 citedByCount "24" @default.
• W1588264401 countsByYear W15882644012012 @default.
• W1588264401 countsByYear W15882644012014 @default.
• W1588264401 countsByYear W15882644012015 @default.
• W1588264401 countsByYear W15882644012016 @default.
• W1588264401 countsByYear W15882644012017 @default.
• W1588264401 countsByYear W15882644012018 @default.
• W1588264401 crossrefType "journal-article" @default.
• W1588264401 hasAuthorship W1588264401A5052448979 @default.
• W1588264401 hasAuthorship W1588264401A5056783795 @default.
• W1588264401 hasAuthorship W1588264401A5079506679 @default.
• W1588264401 hasAuthorship W1588264401A5089344832 @default.
• W1588264401 hasBestOaLocation W15882644011 @default.
• W1588264401 hasConcept C104317684 @default.
• W1588264401 hasConcept C153911025 @default.
• W1588264401 hasConcept C170748874 @default.
• W1588264401 hasConcept C185592680 @default.
• W1588264401 hasConcept C2756471 @default.
• W1588264401 hasConcept C512185932 @default.
• W1588264401 hasConcept C54355233 @default.
• W1588264401 hasConcept C552990157 @default.
• W1588264401 hasConcept C55493867 @default.
• W1588264401 hasConcept C70721500 @default.
• W1588264401 hasConcept C82381507 @default.
• W1588264401 hasConcept C86803240 @default.
• W1588264401 hasConceptScore W1588264401C104317684 @default.
• W1588264401 hasConceptScore W1588264401C153911025 @default.
• W1588264401 hasConceptScore W1588264401C170748874 @default.
• W1588264401 hasConceptScore W1588264401C185592680 @default.
• W1588264401 hasConceptScore W1588264401C2756471 @default.
• W1588264401 hasConceptScore W1588264401C512185932 @default.
• W1588264401 hasConceptScore W1588264401C54355233 @default.
• W1588264401 hasConceptScore W1588264401C552990157 @default.
• W1588264401 hasConceptScore W1588264401C55493867 @default.
• W1588264401 hasConceptScore W1588264401C70721500 @default.
• W1588264401 hasConceptScore W1588264401C82381507 @default.
• W1588264401 hasConceptScore W1588264401C86803240 @default.
• W1588264401 hasIssue "52" @default.
• W1588264401 hasLocation W15882644011 @default.
• W1588264401 hasOpenAccess W1588264401 @default.
• W1588264401 hasPrimaryLocation W15882644011 @default.
• W1588264401 hasRelatedWork W1489886741 @default.
• W1588264401 hasRelatedWork W1974092340 @default.
• W1588264401 hasRelatedWork W1993418869 @default.
• W1588264401 hasRelatedWork W2000232541 @default.
• W1588264401 hasRelatedWork W2016016179 @default.
• W1588264401 hasRelatedWork W2046796049 @default.

• next