Limits...
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.

Show MeSH

Related in: MedlinePlus

Pyrophosphorolysis of TMP-terminated primer. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and mixed with 400 nM α-32P-TTP in the presence of 2.5 mM Mg2+. The reaction was aged for 5 s to permit radiolabeled TTP incorporation. A second mixing event then combined this with varying concentrations of PPi [15 (filled circle), 30 (open circle), 75 (filled square), 200 (open square), 400 (filled triangle) and 1000 (open triangle) μM] in solution with 2.5 mM Mg2+ and 20 μM unlabeled TTP. The unlabeled TTP was included to prevent the undesired incorporation of radiolabeled TTP following the pyrophosphorolysis reaction. Shown is the concentration of the 26-mer plotted as a function of time and the data were analyzed by non-linear regression using a single exponential equation to obtain the observed rate constants at the various PPi concentrations. (B) The observed reaction rates were plotted versus the concentration of PPi used and fitted using Equation (5) to yield a maximum rate of pyrophosphorolysis of 1.4 ± 0.3 s−1 and a Kd of 240 ± 80 μM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175305&req=5

Figure 2: Pyrophosphorolysis of TMP-terminated primer. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and mixed with 400 nM α-32P-TTP in the presence of 2.5 mM Mg2+. The reaction was aged for 5 s to permit radiolabeled TTP incorporation. A second mixing event then combined this with varying concentrations of PPi [15 (filled circle), 30 (open circle), 75 (filled square), 200 (open square), 400 (filled triangle) and 1000 (open triangle) μM] in solution with 2.5 mM Mg2+ and 20 μM unlabeled TTP. The unlabeled TTP was included to prevent the undesired incorporation of radiolabeled TTP following the pyrophosphorolysis reaction. Shown is the concentration of the 26-mer plotted as a function of time and the data were analyzed by non-linear regression using a single exponential equation to obtain the observed rate constants at the various PPi concentrations. (B) The observed reaction rates were plotted versus the concentration of PPi used and fitted using Equation (5) to yield a maximum rate of pyrophosphorolysis of 1.4 ± 0.3 s−1 and a Kd of 240 ± 80 μM.

Mentions: It was clear that the radiolabel must be present in the terminal base rather than the 5′-terminus to make an accurate rate measurement. Therefore, a double mixing experiment was performed where, in the first mixing event, Pol γ/DNA complex (coding for T incorporation) was mixed with α-32P-TTP and allowed to react for 5 s to obtain full incorporation. In the second mixing event this solution was combined with varying concentrations of PPi and 2.5 mM Mg2+, plus 20 μM TTP. The cold TTP was necessary to trap radiolabeled TTP in solution. The results are shown in Figure 2. The time dependence of the disappearance of 26-mer was monophasic and therefore fitted using a single exponential equation to extract the observed rate for each concentration of PPi used. The observed rates of reaction followed a hyperbolic concentration dependence to yield a maximum rate of pyrophosphorolysis of 1.4 ± 0.3 s−1 and a Kd of 240 ± 80 μM. Pol γ was able to catalyze the reverse reaction efficiently, but only using a naturally terminated primer strand. These results were counter to our hypothesis that a substantial reverse rate constant for phosphoryl transfer was the cause of the abnormal kinetics of incorporation using AZT-TP.Figure 2.


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

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

Pyrophosphorolysis of TMP-terminated primer. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and mixed with 400 nM α-32P-TTP in the presence of 2.5 mM Mg2+. The reaction was aged for 5 s to permit radiolabeled TTP incorporation. A second mixing event then combined this with varying concentrations of PPi [15 (filled circle), 30 (open circle), 75 (filled square), 200 (open square), 400 (filled triangle) and 1000 (open triangle) μM] in solution with 2.5 mM Mg2+ and 20 μM unlabeled TTP. The unlabeled TTP was included to prevent the undesired incorporation of radiolabeled TTP following the pyrophosphorolysis reaction. Shown is the concentration of the 26-mer plotted as a function of time and the data were analyzed by non-linear regression using a single exponential equation to obtain the observed rate constants at the various PPi concentrations. (B) The observed reaction rates were plotted versus the concentration of PPi used and fitted using Equation (5) to yield a maximum rate of pyrophosphorolysis of 1.4 ± 0.3 s−1 and a Kd of 240 ± 80 μM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Pyrophosphorolysis of TMP-terminated primer. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and mixed with 400 nM α-32P-TTP in the presence of 2.5 mM Mg2+. The reaction was aged for 5 s to permit radiolabeled TTP incorporation. A second mixing event then combined this with varying concentrations of PPi [15 (filled circle), 30 (open circle), 75 (filled square), 200 (open square), 400 (filled triangle) and 1000 (open triangle) μM] in solution with 2.5 mM Mg2+ and 20 μM unlabeled TTP. The unlabeled TTP was included to prevent the undesired incorporation of radiolabeled TTP following the pyrophosphorolysis reaction. Shown is the concentration of the 26-mer plotted as a function of time and the data were analyzed by non-linear regression using a single exponential equation to obtain the observed rate constants at the various PPi concentrations. (B) The observed reaction rates were plotted versus the concentration of PPi used and fitted using Equation (5) to yield a maximum rate of pyrophosphorolysis of 1.4 ± 0.3 s−1 and a Kd of 240 ± 80 μM.
Mentions: It was clear that the radiolabel must be present in the terminal base rather than the 5′-terminus to make an accurate rate measurement. Therefore, a double mixing experiment was performed where, in the first mixing event, Pol γ/DNA complex (coding for T incorporation) was mixed with α-32P-TTP and allowed to react for 5 s to obtain full incorporation. In the second mixing event this solution was combined with varying concentrations of PPi and 2.5 mM Mg2+, plus 20 μM TTP. The cold TTP was necessary to trap radiolabeled TTP in solution. The results are shown in Figure 2. The time dependence of the disappearance of 26-mer was monophasic and therefore fitted using a single exponential equation to extract the observed rate for each concentration of PPi used. The observed rates of reaction followed a hyperbolic concentration dependence to yield a maximum rate of pyrophosphorolysis of 1.4 ± 0.3 s−1 and a Kd of 240 ± 80 μM. Pol γ was able to catalyze the reverse reaction efficiently, but only using a naturally terminated primer strand. These results were counter to our hypothesis that a substantial reverse rate constant for phosphoryl transfer was the cause of the abnormal kinetics of incorporation using AZT-TP.Figure 2.

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