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ATPase mechanism of the 5'-3' DNA helicase, RecD2: evidence for a pre-hydrolysis conformation change.

Toseland CP, Webb MR - J. Biol. Chem. (2013)

Bottom Line: The data show that a rearrangement linked to Mg(2+) coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA.This is also shown here for the PcrA 3'-5' helicase and so may be a general mechanism governing superfamily 1 helicases.The mechanism accounts for the tight coupling between translocation and ATPase activity.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, London, United Kingdom.

ABSTRACT
The superfamily 1 helicase, RecD2, is a monomeric, bacterial enzyme with a role in DNA repair, but with 5'-3' activity unlike most enzymes from this superfamily. Rate constants were determined for steps within the ATPase cycle of RecD2 in the presence of ssDNA. The fluorescent ATP analog, mantATP (2'(3')-O-(N-methylanthraniloyl)ATP), was used throughout to provide a complete set of rate constants and determine the mechanism of the cycle for a single nucleotide species. Fluorescence stopped-flow measurements were used to determine rate constants for adenosine nucleotide binding and release, quenched-flow measurements were used for the hydrolytic cleavage step, and the fluorescent phosphate biosensor was used for phosphate release kinetics. Some rate constants could also be measured using the natural substrate, ATP, and these suggested a similar mechanism to that obtained with mantATP. The data show that a rearrangement linked to Mg(2+) coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA. This is also shown here for the PcrA 3'-5' helicase and so may be a general mechanism governing superfamily 1 helicases. The mechanism accounts for the tight coupling between translocation and ATPase activity.

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Related in: MedlinePlus

MantATP binding to RecD2·dT20. MantATP at the micromolar concentrations shown was mixed in the stopped flow apparatus with 0.5 μm RecD2 and 2.5 μm dT20 at 20 °C in the buffer described under “Experimental Procedures.” A, individual traces (offset from each other) were fitted to single exponentials (Equation 1), and the dependence of the observed rate constants on concentration was then linear-fitted to Equation 3 (B). The points shown are averages of at least three measurements. The slope gives k+1 as 5.4 ± 0.8 μm−1s−1. The intercept with the ordinate (190 ± 8 s−1) represents k−1 + k+2 (scheme in Fig. 1A). C, shown is the time course after mixing 12.5 μm RecD2 and 15 μm dT20 with 2.5 μm mantATP. The inset shows the initial increase in fluorescence. The complete trace was fitted by a double exponential (Equation 2, dashed line), and the single exponential fit is shown for comparison (dotted line). The first phase, representing two-thirds of the amplitude, had an observed rate constant of 248 ± 7 s−1, and the second phase was 38.4 ± 2.4 s−1. D, traces were fitted to double exponentials, and the dependence of the observed rate constants on concentration was fitted to Equation 3. The observed rate constant for the initial change in fluorescence is linearly dependent on the RecD2 concentration (unfilled circles). After a linear fit to Equation 3, the second order association rate constant was 5.7 ± 0.7 μm−1s−1, and the intercept was 158 ± 9 s−1. The observed rate constants of the second increase in fluorescence (filled circles) were independent of RecD2 concentration at 39 s−1.
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Figure 3: MantATP binding to RecD2·dT20. MantATP at the micromolar concentrations shown was mixed in the stopped flow apparatus with 0.5 μm RecD2 and 2.5 μm dT20 at 20 °C in the buffer described under “Experimental Procedures.” A, individual traces (offset from each other) were fitted to single exponentials (Equation 1), and the dependence of the observed rate constants on concentration was then linear-fitted to Equation 3 (B). The points shown are averages of at least three measurements. The slope gives k+1 as 5.4 ± 0.8 μm−1s−1. The intercept with the ordinate (190 ± 8 s−1) represents k−1 + k+2 (scheme in Fig. 1A). C, shown is the time course after mixing 12.5 μm RecD2 and 15 μm dT20 with 2.5 μm mantATP. The inset shows the initial increase in fluorescence. The complete trace was fitted by a double exponential (Equation 2, dashed line), and the single exponential fit is shown for comparison (dotted line). The first phase, representing two-thirds of the amplitude, had an observed rate constant of 248 ± 7 s−1, and the second phase was 38.4 ± 2.4 s−1. D, traces were fitted to double exponentials, and the dependence of the observed rate constants on concentration was fitted to Equation 3. The observed rate constant for the initial change in fluorescence is linearly dependent on the RecD2 concentration (unfilled circles). After a linear fit to Equation 3, the second order association rate constant was 5.7 ± 0.7 μm−1s−1, and the intercept was 158 ± 9 s−1. The observed rate constants of the second increase in fluorescence (filled circles) were independent of RecD2 concentration at 39 s−1.

Mentions: The kinetics for mantATP binding to RecD2·dT20 were measured under pseudo-first order conditions with mantATP in large excess over the protein. Using the stopped-flow apparatus, several concentrations of mantATP were rapidly mixed with the RecD2·dT20 complex, and fluorescence was followed with time (Fig. 3A). The increase in fluorescence was fitted by a single exponential. The observed rate constants were linearly dependent on mantATP concentration (Fig. 3B), giving a second order association rate constant of 5.4 μm−1s−1 from the slope. The intercept with the ordinate (190 s−1) is the sum of rate constants controlling breakdown of the bound mantATP, which include both the triphosphate dissociation and hydrolysis.


ATPase mechanism of the 5'-3' DNA helicase, RecD2: evidence for a pre-hydrolysis conformation change.

Toseland CP, Webb MR - J. Biol. Chem. (2013)

MantATP binding to RecD2·dT20. MantATP at the micromolar concentrations shown was mixed in the stopped flow apparatus with 0.5 μm RecD2 and 2.5 μm dT20 at 20 °C in the buffer described under “Experimental Procedures.” A, individual traces (offset from each other) were fitted to single exponentials (Equation 1), and the dependence of the observed rate constants on concentration was then linear-fitted to Equation 3 (B). The points shown are averages of at least three measurements. The slope gives k+1 as 5.4 ± 0.8 μm−1s−1. The intercept with the ordinate (190 ± 8 s−1) represents k−1 + k+2 (scheme in Fig. 1A). C, shown is the time course after mixing 12.5 μm RecD2 and 15 μm dT20 with 2.5 μm mantATP. The inset shows the initial increase in fluorescence. The complete trace was fitted by a double exponential (Equation 2, dashed line), and the single exponential fit is shown for comparison (dotted line). The first phase, representing two-thirds of the amplitude, had an observed rate constant of 248 ± 7 s−1, and the second phase was 38.4 ± 2.4 s−1. D, traces were fitted to double exponentials, and the dependence of the observed rate constants on concentration was fitted to Equation 3. The observed rate constant for the initial change in fluorescence is linearly dependent on the RecD2 concentration (unfilled circles). After a linear fit to Equation 3, the second order association rate constant was 5.7 ± 0.7 μm−1s−1, and the intercept was 158 ± 9 s−1. The observed rate constants of the second increase in fluorescence (filled circles) were independent of RecD2 concentration at 39 s−1.
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Figure 3: MantATP binding to RecD2·dT20. MantATP at the micromolar concentrations shown was mixed in the stopped flow apparatus with 0.5 μm RecD2 and 2.5 μm dT20 at 20 °C in the buffer described under “Experimental Procedures.” A, individual traces (offset from each other) were fitted to single exponentials (Equation 1), and the dependence of the observed rate constants on concentration was then linear-fitted to Equation 3 (B). The points shown are averages of at least three measurements. The slope gives k+1 as 5.4 ± 0.8 μm−1s−1. The intercept with the ordinate (190 ± 8 s−1) represents k−1 + k+2 (scheme in Fig. 1A). C, shown is the time course after mixing 12.5 μm RecD2 and 15 μm dT20 with 2.5 μm mantATP. The inset shows the initial increase in fluorescence. The complete trace was fitted by a double exponential (Equation 2, dashed line), and the single exponential fit is shown for comparison (dotted line). The first phase, representing two-thirds of the amplitude, had an observed rate constant of 248 ± 7 s−1, and the second phase was 38.4 ± 2.4 s−1. D, traces were fitted to double exponentials, and the dependence of the observed rate constants on concentration was fitted to Equation 3. The observed rate constant for the initial change in fluorescence is linearly dependent on the RecD2 concentration (unfilled circles). After a linear fit to Equation 3, the second order association rate constant was 5.7 ± 0.7 μm−1s−1, and the intercept was 158 ± 9 s−1. The observed rate constants of the second increase in fluorescence (filled circles) were independent of RecD2 concentration at 39 s−1.
Mentions: The kinetics for mantATP binding to RecD2·dT20 were measured under pseudo-first order conditions with mantATP in large excess over the protein. Using the stopped-flow apparatus, several concentrations of mantATP were rapidly mixed with the RecD2·dT20 complex, and fluorescence was followed with time (Fig. 3A). The increase in fluorescence was fitted by a single exponential. The observed rate constants were linearly dependent on mantATP concentration (Fig. 3B), giving a second order association rate constant of 5.4 μm−1s−1 from the slope. The intercept with the ordinate (190 s−1) is the sum of rate constants controlling breakdown of the bound mantATP, which include both the triphosphate dissociation and hydrolysis.

Bottom Line: The data show that a rearrangement linked to Mg(2+) coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA.This is also shown here for the PcrA 3'-5' helicase and so may be a general mechanism governing superfamily 1 helicases.The mechanism accounts for the tight coupling between translocation and ATPase activity.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, London, United Kingdom.

ABSTRACT
The superfamily 1 helicase, RecD2, is a monomeric, bacterial enzyme with a role in DNA repair, but with 5'-3' activity unlike most enzymes from this superfamily. Rate constants were determined for steps within the ATPase cycle of RecD2 in the presence of ssDNA. The fluorescent ATP analog, mantATP (2'(3')-O-(N-methylanthraniloyl)ATP), was used throughout to provide a complete set of rate constants and determine the mechanism of the cycle for a single nucleotide species. Fluorescence stopped-flow measurements were used to determine rate constants for adenosine nucleotide binding and release, quenched-flow measurements were used for the hydrolytic cleavage step, and the fluorescent phosphate biosensor was used for phosphate release kinetics. Some rate constants could also be measured using the natural substrate, ATP, and these suggested a similar mechanism to that obtained with mantATP. The data show that a rearrangement linked to Mg(2+) coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA. This is also shown here for the PcrA 3'-5' helicase and so may be a general mechanism governing superfamily 1 helicases. The mechanism accounts for the tight coupling between translocation and ATPase activity.

Show MeSH
Related in: MedlinePlus