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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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Comparison of PPi release following incorporation using TTP or AZT-TP. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated for 5 min with 2.5 mM Mg2+, 90 nM 25/45-mer DNA, 1.5 μM E. coli PBP mutant labeled at Cys197 with the fluorescent compound MDCC, 100 μM 7-methylguanosine, 0.02 U/ml purine nucleotide phosphorylase and 0.005 U/μl yeast inorganic PPase and then rapidly mixed in the stopped-flow apparatus at 20 ° C with 2.5 mM Mg2+, 50 μM TTP, 100 μM 7-methylguanosine, and 0.02 U/ml purine nucleotide phosphorylase. PPi dissociation was monitored by observing the change in fluorescence that results from Pi binding to the MDCC-labeled PBP (smooth line). Shown also are quench-flow data from a parallel experiment monitoring the DNAn+1 formation using a radiolabeled primer strand (filled circle). (B) Exonuclease-deficient holoenzyme (1 μM) was preincubated with 900 nM 25/45-mer DNA and mixed with 300 μM γ-32P-labeled AZT-TP and 2.5 mM Mg2+ to start the reaction. The amount of PPi (or PPi in equilibrium with AZT-TP) that remained associated with Pol γ as a function of time is shown on the plot. The data were fitted to a double exponential equation to obtain an observed fast phase rate and amplitude of 1.2 ± 0.3 min−1 and 700 ± 70 nM, respectively. The observed rate and amplitude of the slow phase were 0.007 ± 0.002 min−1 and 700 ± 50 nM, respectively.
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Figure 3: Comparison of PPi release following incorporation using TTP or AZT-TP. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated for 5 min with 2.5 mM Mg2+, 90 nM 25/45-mer DNA, 1.5 μM E. coli PBP mutant labeled at Cys197 with the fluorescent compound MDCC, 100 μM 7-methylguanosine, 0.02 U/ml purine nucleotide phosphorylase and 0.005 U/μl yeast inorganic PPase and then rapidly mixed in the stopped-flow apparatus at 20 ° C with 2.5 mM Mg2+, 50 μM TTP, 100 μM 7-methylguanosine, and 0.02 U/ml purine nucleotide phosphorylase. PPi dissociation was monitored by observing the change in fluorescence that results from Pi binding to the MDCC-labeled PBP (smooth line). Shown also are quench-flow data from a parallel experiment monitoring the DNAn+1 formation using a radiolabeled primer strand (filled circle). (B) Exonuclease-deficient holoenzyme (1 μM) was preincubated with 900 nM 25/45-mer DNA and mixed with 300 μM γ-32P-labeled AZT-TP and 2.5 mM Mg2+ to start the reaction. The amount of PPi (or PPi in equilibrium with AZT-TP) that remained associated with Pol γ as a function of time is shown on the plot. The data were fitted to a double exponential equation to obtain an observed fast phase rate and amplitude of 1.2 ± 0.3 min−1 and 700 ± 70 nM, respectively. The observed rate and amplitude of the slow phase were 0.007 ± 0.002 min−1 and 700 ± 50 nM, respectively.

Mentions: The next obvious question was regarding the kinetics of PPi release following the chemical reaction. It appeared from the previous experiments that free PPi had no influence on the reaction amplitude. The data seemed to be suggesting that the dissociation of PPi was not occurring rapidly following the formation of product. Therefore, it was of interest to measure the rate of PPi release during the forward reaction. An assay was developed using TTP in order to be sure that it was capable of quantifying PPi dissociation accurately. The fundamental component in obtaining a signal was the mutant MDCC-labeled phosphate-binding protein (MDCC-PBP) that was developed for monitoring the release of Pi (24). This protein exhibits a rapid and large fluorescence increase upon binding Pi. However, PPi does not bind and induce a fluorescence change, so in order for this assay to function, yeast inorganic PPase was included. Shown in Figure 3A (smooth line) is the fluorescent signal collected when an incorporation reaction was performed under single turnover conditions with a saturating concentration of TTP. To compare this to the kinetics of the incorporation reaction, an experiment was performed in the chemical quench flow with a radiolabeled primer strand. The results are plotted along with the stopped-flow data in Figure 3A. The quench-flow data overlay the fluorescence trace even though one is measuring PPi release and the other is measuring the formation of DNAn+1. In addition, there is no observable lag phase in the stopped-flow fluorescence data. These results indicate that the rate constant for PPi dissociation must be at least five times greater than the maximum rate of incorporation using TTP (∼25 s−1 at 20 C). It is quite possible that a conformational change of the protein actually limits the rate of PPi dissociation, but this data alone does not provide such evidence, but sets a lower limit on the rate of PPi release of approximately five times the observed rate, 125 s−1.Figure 3.


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

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

Comparison of PPi release following incorporation using TTP or AZT-TP. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated for 5 min with 2.5 mM Mg2+, 90 nM 25/45-mer DNA, 1.5 μM E. coli PBP mutant labeled at Cys197 with the fluorescent compound MDCC, 100 μM 7-methylguanosine, 0.02 U/ml purine nucleotide phosphorylase and 0.005 U/μl yeast inorganic PPase and then rapidly mixed in the stopped-flow apparatus at 20 ° C with 2.5 mM Mg2+, 50 μM TTP, 100 μM 7-methylguanosine, and 0.02 U/ml purine nucleotide phosphorylase. PPi dissociation was monitored by observing the change in fluorescence that results from Pi binding to the MDCC-labeled PBP (smooth line). Shown also are quench-flow data from a parallel experiment monitoring the DNAn+1 formation using a radiolabeled primer strand (filled circle). (B) Exonuclease-deficient holoenzyme (1 μM) was preincubated with 900 nM 25/45-mer DNA and mixed with 300 μM γ-32P-labeled AZT-TP and 2.5 mM Mg2+ to start the reaction. The amount of PPi (or PPi in equilibrium with AZT-TP) that remained associated with Pol γ as a function of time is shown on the plot. The data were fitted to a double exponential equation to obtain an observed fast phase rate and amplitude of 1.2 ± 0.3 min−1 and 700 ± 70 nM, respectively. The observed rate and amplitude of the slow phase were 0.007 ± 0.002 min−1 and 700 ± 50 nM, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175305&req=5

Figure 3: Comparison of PPi release following incorporation using TTP or AZT-TP. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated for 5 min with 2.5 mM Mg2+, 90 nM 25/45-mer DNA, 1.5 μM E. coli PBP mutant labeled at Cys197 with the fluorescent compound MDCC, 100 μM 7-methylguanosine, 0.02 U/ml purine nucleotide phosphorylase and 0.005 U/μl yeast inorganic PPase and then rapidly mixed in the stopped-flow apparatus at 20 ° C with 2.5 mM Mg2+, 50 μM TTP, 100 μM 7-methylguanosine, and 0.02 U/ml purine nucleotide phosphorylase. PPi dissociation was monitored by observing the change in fluorescence that results from Pi binding to the MDCC-labeled PBP (smooth line). Shown also are quench-flow data from a parallel experiment monitoring the DNAn+1 formation using a radiolabeled primer strand (filled circle). (B) Exonuclease-deficient holoenzyme (1 μM) was preincubated with 900 nM 25/45-mer DNA and mixed with 300 μM γ-32P-labeled AZT-TP and 2.5 mM Mg2+ to start the reaction. The amount of PPi (or PPi in equilibrium with AZT-TP) that remained associated with Pol γ as a function of time is shown on the plot. The data were fitted to a double exponential equation to obtain an observed fast phase rate and amplitude of 1.2 ± 0.3 min−1 and 700 ± 70 nM, respectively. The observed rate and amplitude of the slow phase were 0.007 ± 0.002 min−1 and 700 ± 50 nM, respectively.
Mentions: The next obvious question was regarding the kinetics of PPi release following the chemical reaction. It appeared from the previous experiments that free PPi had no influence on the reaction amplitude. The data seemed to be suggesting that the dissociation of PPi was not occurring rapidly following the formation of product. Therefore, it was of interest to measure the rate of PPi release during the forward reaction. An assay was developed using TTP in order to be sure that it was capable of quantifying PPi dissociation accurately. The fundamental component in obtaining a signal was the mutant MDCC-labeled phosphate-binding protein (MDCC-PBP) that was developed for monitoring the release of Pi (24). This protein exhibits a rapid and large fluorescence increase upon binding Pi. However, PPi does not bind and induce a fluorescence change, so in order for this assay to function, yeast inorganic PPase was included. Shown in Figure 3A (smooth line) is the fluorescent signal collected when an incorporation reaction was performed under single turnover conditions with a saturating concentration of TTP. To compare this to the kinetics of the incorporation reaction, an experiment was performed in the chemical quench flow with a radiolabeled primer strand. The results are plotted along with the stopped-flow data in Figure 3A. The quench-flow data overlay the fluorescence trace even though one is measuring PPi release and the other is measuring the formation of DNAn+1. In addition, there is no observable lag phase in the stopped-flow fluorescence data. These results indicate that the rate constant for PPi dissociation must be at least five times greater than the maximum rate of incorporation using TTP (∼25 s−1 at 20 C). It is quite possible that a conformational change of the protein actually limits the rate of PPi dissociation, but this data alone does not provide such evidence, but sets a lower limit on the rate of PPi release of approximately five times the observed rate, 125 s−1.Figure 3.

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