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A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase.

Hanes JW, Johnson KA - Nucleic Acids Res. (2007)

Bottom Line: The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked.This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/K(m) (7 x 10(-6) microM(-1) s(-1)).Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry & Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712, USA.

ABSTRACT
Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5'-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s(-1)) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 microM) and chemistry forward and reverse reactions, 0.38 and 0.22 s(-1), respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/K(m) (7 x 10(-6) microM(-1) s(-1)). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena.

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Kinetics of incorporation using azide-substituted analogs. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+ and various concentrations of AZA-TP [50 (open triangle), 100 (filled square), 200 (open square), 300 (filled circle) and 400 μM (open triangle)]. Each data set was fitted using a double exponential equation. (B) The amplitude for fast phase of each reaction was plotted as a function of AZA-TP concentration (filled circle). A fit of the data to Equation (2). yields an apparent Kd of 800 ± 120 μM and a maximum amplitude of 65 ± 8 nM. The observed rate of the fast phase of each reaction was also plotted as a function AZA-TP concentration (open square). (C) Single turnover reaction performed using AZC-TP [10 (open triangle), 35 (filled square), 65 (open square), 150 (filled circle) and 300 μM (open circle)]. (D) The amplitude was plotted as a function of AZC-TP concentration (filled circle). A fit of the data predicts an apparent Kd of 96 ± 6 μM and a maximum amplitude of 64 ± 2 nM. The observed rate of the fast phase of each reaction was also plotted (open square). (E) Single turnover reaction using AZG-TP [50 (filled triangle), 100 (open triangle), 200 (filled square), 325 (open square), 450 (filled circle) and 600 μM (open square)]. Each data set was fitted using a single exponential equation. (F) The amplitude was plotted as a function of AZG-TP concentration (filled circle) to define an apparent Kd of 50 ± 10 μM and a maximum amplitude of 81 ± 3 nM. The observed rate of each reaction was also plotted and fitted by linear regression to obtain a slope of (7.5 ± 0.5) × 10−5 μM−1 min−1and a Y-intercept of 0.028 ± 0.002 min−1 (open square).
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Figure 6: Kinetics of incorporation using azide-substituted analogs. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+ and various concentrations of AZA-TP [50 (open triangle), 100 (filled square), 200 (open square), 300 (filled circle) and 400 μM (open triangle)]. Each data set was fitted using a double exponential equation. (B) The amplitude for fast phase of each reaction was plotted as a function of AZA-TP concentration (filled circle). A fit of the data to Equation (2). yields an apparent Kd of 800 ± 120 μM and a maximum amplitude of 65 ± 8 nM. The observed rate of the fast phase of each reaction was also plotted as a function AZA-TP concentration (open square). (C) Single turnover reaction performed using AZC-TP [10 (open triangle), 35 (filled square), 65 (open square), 150 (filled circle) and 300 μM (open circle)]. (D) The amplitude was plotted as a function of AZC-TP concentration (filled circle). A fit of the data predicts an apparent Kd of 96 ± 6 μM and a maximum amplitude of 64 ± 2 nM. The observed rate of the fast phase of each reaction was also plotted (open square). (E) Single turnover reaction using AZG-TP [50 (filled triangle), 100 (open triangle), 200 (filled square), 325 (open square), 450 (filled circle) and 600 μM (open square)]. Each data set was fitted using a single exponential equation. (F) The amplitude was plotted as a function of AZG-TP concentration (filled circle) to define an apparent Kd of 50 ± 10 μM and a maximum amplitude of 81 ± 3 nM. The observed rate of each reaction was also plotted and fitted by linear regression to obtain a slope of (7.5 ± 0.5) × 10−5 μM−1 min−1and a Y-intercept of 0.028 ± 0.002 min−1 (open square).

Mentions: The only structural difference between TTP and AZT-TP is that the normal 3′-hydroxyl has been substituted with an azide group. Therefore, the complex kinetics of incorporation is a direct consequence of the azide substitution. In order to determine whether the effect of the azide is general, the other three nucleoside analogs were examined. The single nucleotide incorporation reactions for AZA-TP (dATP analog), AZC-TP (dCTP analog) and AZG-TP (dGTP analog) were carried out in the same fashion as that detailed for AZT-TP and the data obtained are shown in Figure 6. The kinetics of incorporation strongly resembled that of AZT-TP, even though the data collected with AZA-TP and AZC-TP were biphasic. The amplitude of the fast phase of the reaction for both analogs was clearly dependent on the concentration of substrate in solution, while the rate of the fast phase was not (Figure 6A–D). The incorporation assay using AZG-TP, on the other hand, was much slower and monophasic, although the reaction amplitude still appeared to vary with concentration (Figure 6E and F). One significant difference between AZG-TP and the other three analogs was that the rate clearly increased linearly over the concentration range examined.Figure 6.


A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase.

Hanes JW, Johnson KA - Nucleic Acids Res. (2007)

Kinetics of incorporation using azide-substituted analogs. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+ and various concentrations of AZA-TP [50 (open triangle), 100 (filled square), 200 (open square), 300 (filled circle) and 400 μM (open triangle)]. Each data set was fitted using a double exponential equation. (B) The amplitude for fast phase of each reaction was plotted as a function of AZA-TP concentration (filled circle). A fit of the data to Equation (2). yields an apparent Kd of 800 ± 120 μM and a maximum amplitude of 65 ± 8 nM. The observed rate of the fast phase of each reaction was also plotted as a function AZA-TP concentration (open square). (C) Single turnover reaction performed using AZC-TP [10 (open triangle), 35 (filled square), 65 (open square), 150 (filled circle) and 300 μM (open circle)]. (D) The amplitude was plotted as a function of AZC-TP concentration (filled circle). A fit of the data predicts an apparent Kd of 96 ± 6 μM and a maximum amplitude of 64 ± 2 nM. The observed rate of the fast phase of each reaction was also plotted (open square). (E) Single turnover reaction using AZG-TP [50 (filled triangle), 100 (open triangle), 200 (filled square), 325 (open square), 450 (filled circle) and 600 μM (open square)]. Each data set was fitted using a single exponential equation. (F) The amplitude was plotted as a function of AZG-TP concentration (filled circle) to define an apparent Kd of 50 ± 10 μM and a maximum amplitude of 81 ± 3 nM. The observed rate of each reaction was also plotted and fitted by linear regression to obtain a slope of (7.5 ± 0.5) × 10−5 μM−1 min−1and a Y-intercept of 0.028 ± 0.002 min−1 (open square).
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Figure 6: Kinetics of incorporation using azide-substituted analogs. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and then rapidly mixed with Mg2+ and various concentrations of AZA-TP [50 (open triangle), 100 (filled square), 200 (open square), 300 (filled circle) and 400 μM (open triangle)]. Each data set was fitted using a double exponential equation. (B) The amplitude for fast phase of each reaction was plotted as a function of AZA-TP concentration (filled circle). A fit of the data to Equation (2). yields an apparent Kd of 800 ± 120 μM and a maximum amplitude of 65 ± 8 nM. The observed rate of the fast phase of each reaction was also plotted as a function AZA-TP concentration (open square). (C) Single turnover reaction performed using AZC-TP [10 (open triangle), 35 (filled square), 65 (open square), 150 (filled circle) and 300 μM (open circle)]. (D) The amplitude was plotted as a function of AZC-TP concentration (filled circle). A fit of the data predicts an apparent Kd of 96 ± 6 μM and a maximum amplitude of 64 ± 2 nM. The observed rate of the fast phase of each reaction was also plotted (open square). (E) Single turnover reaction using AZG-TP [50 (filled triangle), 100 (open triangle), 200 (filled square), 325 (open square), 450 (filled circle) and 600 μM (open square)]. Each data set was fitted using a single exponential equation. (F) The amplitude was plotted as a function of AZG-TP concentration (filled circle) to define an apparent Kd of 50 ± 10 μM and a maximum amplitude of 81 ± 3 nM. The observed rate of each reaction was also plotted and fitted by linear regression to obtain a slope of (7.5 ± 0.5) × 10−5 μM−1 min−1and a Y-intercept of 0.028 ± 0.002 min−1 (open square).
Mentions: The only structural difference between TTP and AZT-TP is that the normal 3′-hydroxyl has been substituted with an azide group. Therefore, the complex kinetics of incorporation is a direct consequence of the azide substitution. In order to determine whether the effect of the azide is general, the other three nucleoside analogs were examined. The single nucleotide incorporation reactions for AZA-TP (dATP analog), AZC-TP (dCTP analog) and AZG-TP (dGTP analog) were carried out in the same fashion as that detailed for AZT-TP and the data obtained are shown in Figure 6. The kinetics of incorporation strongly resembled that of AZT-TP, even though the data collected with AZA-TP and AZC-TP were biphasic. The amplitude of the fast phase of the reaction for both analogs was clearly dependent on the concentration of substrate in solution, while the rate of the fast phase was not (Figure 6A–D). The incorporation assay using AZG-TP, on the other hand, was much slower and monophasic, although the reaction amplitude still appeared to vary with concentration (Figure 6E and F). One significant difference between AZG-TP and the other three analogs was that the rate clearly increased linearly over the concentration range examined.Figure 6.

Bottom Line: The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked.This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/K(m) (7 x 10(-6) microM(-1) s(-1)).Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry & Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712, USA.

ABSTRACT
Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5'-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s(-1)) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 microM) and chemistry forward and reverse reactions, 0.38 and 0.22 s(-1), respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/K(m) (7 x 10(-6) microM(-1) s(-1)). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena.

Show MeSH
Related in: MedlinePlus