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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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Kinetic measurement of mantATP with excess of RecD2·dT20: binding, hydrolysis, and Pi release. Time course of mant fluorescence (solid line), mantADP formation (circles), and Pi release (dashed line) is shown. All measurements were at 2.5 μm mantATP, 12.5 μm RecD2, 15 μm dT20, and 10 μm MDCC-PBP (for Pi measurement) and were carried out under the conditions of Fig. 3 as described under “Experimental Procedures.” The time courses were simulated (dotted lines), based upon a global model for a single turnover of mantATP based on the scheme in Fig. 1B, as described under “Results.” The simulated mant fluorescence and cleavage time course are in the main panel, and the Pi simulation with the experimental data is in the inset. This gave an observed first order rate constant for mantATP binding ([RecD2] × k+1a) at 200 s−1 followed by the conformation change (k+1b) at 54 s−1, hydrolysis (k+2) at >300 s−1; Pi release (k+3) was fast (>300 s−1).
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Figure 5: Kinetic measurement of mantATP with excess of RecD2·dT20: binding, hydrolysis, and Pi release. Time course of mant fluorescence (solid line), mantADP formation (circles), and Pi release (dashed line) is shown. All measurements were at 2.5 μm mantATP, 12.5 μm RecD2, 15 μm dT20, and 10 μm MDCC-PBP (for Pi measurement) and were carried out under the conditions of Fig. 3 as described under “Experimental Procedures.” The time courses were simulated (dotted lines), based upon a global model for a single turnover of mantATP based on the scheme in Fig. 1B, as described under “Results.” The simulated mant fluorescence and cleavage time course are in the main panel, and the Pi simulation with the experimental data is in the inset. This gave an observed first order rate constant for mantATP binding ([RecD2] × k+1a) at 200 s−1 followed by the conformation change (k+1b) at 54 s−1, hydrolysis (k+2) at >300 s−1; Pi release (k+3) was fast (>300 s−1).

Mentions: The single-turnover hydrolytic cleavage was compared with the mantATP fluorescence measurements under similar conditions of excess RecD2·dT20 over mantATP, as described above. In this way binding, hydrolytic cleavage, and product release can be directly related to each other. Quenched-flow measurements allowed the formation of diphosphate to be monitored with high time resolution (Fig. 5).


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

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

Kinetic measurement of mantATP with excess of RecD2·dT20: binding, hydrolysis, and Pi release. Time course of mant fluorescence (solid line), mantADP formation (circles), and Pi release (dashed line) is shown. All measurements were at 2.5 μm mantATP, 12.5 μm RecD2, 15 μm dT20, and 10 μm MDCC-PBP (for Pi measurement) and were carried out under the conditions of Fig. 3 as described under “Experimental Procedures.” The time courses were simulated (dotted lines), based upon a global model for a single turnover of mantATP based on the scheme in Fig. 1B, as described under “Results.” The simulated mant fluorescence and cleavage time course are in the main panel, and the Pi simulation with the experimental data is in the inset. This gave an observed first order rate constant for mantATP binding ([RecD2] × k+1a) at 200 s−1 followed by the conformation change (k+1b) at 54 s−1, hydrolysis (k+2) at >300 s−1; Pi release (k+3) was fast (>300 s−1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757182&req=5

Figure 5: Kinetic measurement of mantATP with excess of RecD2·dT20: binding, hydrolysis, and Pi release. Time course of mant fluorescence (solid line), mantADP formation (circles), and Pi release (dashed line) is shown. All measurements were at 2.5 μm mantATP, 12.5 μm RecD2, 15 μm dT20, and 10 μm MDCC-PBP (for Pi measurement) and were carried out under the conditions of Fig. 3 as described under “Experimental Procedures.” The time courses were simulated (dotted lines), based upon a global model for a single turnover of mantATP based on the scheme in Fig. 1B, as described under “Results.” The simulated mant fluorescence and cleavage time course are in the main panel, and the Pi simulation with the experimental data is in the inset. This gave an observed first order rate constant for mantATP binding ([RecD2] × k+1a) at 200 s−1 followed by the conformation change (k+1b) at 54 s−1, hydrolysis (k+2) at >300 s−1; Pi release (k+3) was fast (>300 s−1).
Mentions: The single-turnover hydrolytic cleavage was compared with the mantATP fluorescence measurements under similar conditions of excess RecD2·dT20 over mantATP, as described above. In this way binding, hydrolytic cleavage, and product release can be directly related to each other. Quenched-flow measurements allowed the formation of diphosphate to be monitored with high time resolution (Fig. 5).

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