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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

PcrA helicase ATPase kinetics with deac-aminoATP: binding, hydrolysis, and Pi release.A, shown is PcrA·dT20 binding deac-aminoADP. Deac-aminoADP at various concentrations was mixed in the stopped flow apparatus with 0.5 μm PcrA and 2.5 μm dT20. Traces were fitted by single exponentials. The observed rate constants are shown as a function of concentration, and the best linear fit (Equation 3) gives an association rate constant of 4.2 ± 0.3 μm−1 s−1 and a dissociation rate constant of 7.3 ± 2.2 s−1, resulting in a dissociation constant of 1.7 μm. B, shown is deac-aminoATP binding with excess RecD2. The concentrations were 2 μm deac-aminoATP, 8 μm PcrA, 10 μm dT20. The time course of fluorescence (solid line) was fitted by a double exponential. The initial increase in fluorescence had a rate constant of 130 ± 3 s−1, and this was followed by a slower change in fluorescence with a rate constant of 26.2 ± 0.9 s−1. C, shown is deac-aminoATP cleavage and Pi release. The concentrations were as for panel B. The binding traces were repeated from panel B. Deac-aminoADP formation (circles) was measured using quench flow and HPLC. Pi release (dashed line) was measured in the presence of 10 μm rhodamine-PBP. The simulation in Fig. 5 was applied to fit the dataset. The simulated binding and cleavage are the dashed line in the main panel. The simulated Pi release is in the inset. This gave the observed first order rate constant for deac-aminoATP binding (equivalent to [PcrA] × k+1a) at 100 s−1 followed by a conformation change (equivalent to k+1b) at 29 s−1, hydrolytic cleavage at >100 s−1 and Pi release at >300 s−1.
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Figure 10: PcrA helicase ATPase kinetics with deac-aminoATP: binding, hydrolysis, and Pi release.A, shown is PcrA·dT20 binding deac-aminoADP. Deac-aminoADP at various concentrations was mixed in the stopped flow apparatus with 0.5 μm PcrA and 2.5 μm dT20. Traces were fitted by single exponentials. The observed rate constants are shown as a function of concentration, and the best linear fit (Equation 3) gives an association rate constant of 4.2 ± 0.3 μm−1 s−1 and a dissociation rate constant of 7.3 ± 2.2 s−1, resulting in a dissociation constant of 1.7 μm. B, shown is deac-aminoATP binding with excess RecD2. The concentrations were 2 μm deac-aminoATP, 8 μm PcrA, 10 μm dT20. The time course of fluorescence (solid line) was fitted by a double exponential. The initial increase in fluorescence had a rate constant of 130 ± 3 s−1, and this was followed by a slower change in fluorescence with a rate constant of 26.2 ± 0.9 s−1. C, shown is deac-aminoATP cleavage and Pi release. The concentrations were as for panel B. The binding traces were repeated from panel B. Deac-aminoADP formation (circles) was measured using quench flow and HPLC. Pi release (dashed line) was measured in the presence of 10 μm rhodamine-PBP. The simulation in Fig. 5 was applied to fit the dataset. The simulated binding and cleavage are the dashed line in the main panel. The simulated Pi release is in the inset. This gave the observed first order rate constant for deac-aminoATP binding (equivalent to [PcrA] × k+1a) at 100 s−1 followed by a conformation change (equivalent to k+1b) at 29 s−1, hydrolytic cleavage at >100 s−1 and Pi release at >300 s−1.

Mentions: To test this hypothesis, a single turnover measurement, equivalent to Fig. 5, was done with PcrA helicase but using the fluorescent ATP analog, deac-aminoATP (16). Deac-aminoADP has a significantly tighter interaction with PcrA with a Kd of 1.7 μm (Fig. 10A), so almost all the diphosphate should remain bound during the single turnover experiment. After mixing excess PcrA·dT20 with deac-aminoATP, there was a biphasic increase in fluorescence (Fig. 10B). The initial increase in fluorescence, which accounted for 75% of the overall fluorescence change, had a rate constant of 130 s−1. This was followed by a slower change in fluorescence with a rate constant of 26 s−1. The first phase in fluorescence is presumably binding, whereas the second change could be due to a pre-cleavage conformation change. This was supported by measurements of hydrolysis by quench-flow and Pi release using a phosphate biosensor under the same conditions (Fig. 10B). To show the similarity in the mechanism between RecD2 and PcrA, the same model, used to fit data in Fig. 5, was applied to fit this dataset (Fig. 10C). This gave deac-aminoATP binding with a first order rate constant (equivalent to [PcrA] × k+1a; Fig. 1B) of 100 s−1 followed by a conformation change binding (equivalent to k+1b) of 29 s−1, hydrolytic cleavage at >100 s−1, and Pi release at >300 s−1.


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

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

PcrA helicase ATPase kinetics with deac-aminoATP: binding, hydrolysis, and Pi release.A, shown is PcrA·dT20 binding deac-aminoADP. Deac-aminoADP at various concentrations was mixed in the stopped flow apparatus with 0.5 μm PcrA and 2.5 μm dT20. Traces were fitted by single exponentials. The observed rate constants are shown as a function of concentration, and the best linear fit (Equation 3) gives an association rate constant of 4.2 ± 0.3 μm−1 s−1 and a dissociation rate constant of 7.3 ± 2.2 s−1, resulting in a dissociation constant of 1.7 μm. B, shown is deac-aminoATP binding with excess RecD2. The concentrations were 2 μm deac-aminoATP, 8 μm PcrA, 10 μm dT20. The time course of fluorescence (solid line) was fitted by a double exponential. The initial increase in fluorescence had a rate constant of 130 ± 3 s−1, and this was followed by a slower change in fluorescence with a rate constant of 26.2 ± 0.9 s−1. C, shown is deac-aminoATP cleavage and Pi release. The concentrations were as for panel B. The binding traces were repeated from panel B. Deac-aminoADP formation (circles) was measured using quench flow and HPLC. Pi release (dashed line) was measured in the presence of 10 μm rhodamine-PBP. The simulation in Fig. 5 was applied to fit the dataset. The simulated binding and cleavage are the dashed line in the main panel. The simulated Pi release is in the inset. This gave the observed first order rate constant for deac-aminoATP binding (equivalent to [PcrA] × k+1a) at 100 s−1 followed by a conformation change (equivalent to k+1b) at 29 s−1, hydrolytic cleavage at >100 s−1 and Pi release at >300 s−1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 10: PcrA helicase ATPase kinetics with deac-aminoATP: binding, hydrolysis, and Pi release.A, shown is PcrA·dT20 binding deac-aminoADP. Deac-aminoADP at various concentrations was mixed in the stopped flow apparatus with 0.5 μm PcrA and 2.5 μm dT20. Traces were fitted by single exponentials. The observed rate constants are shown as a function of concentration, and the best linear fit (Equation 3) gives an association rate constant of 4.2 ± 0.3 μm−1 s−1 and a dissociation rate constant of 7.3 ± 2.2 s−1, resulting in a dissociation constant of 1.7 μm. B, shown is deac-aminoATP binding with excess RecD2. The concentrations were 2 μm deac-aminoATP, 8 μm PcrA, 10 μm dT20. The time course of fluorescence (solid line) was fitted by a double exponential. The initial increase in fluorescence had a rate constant of 130 ± 3 s−1, and this was followed by a slower change in fluorescence with a rate constant of 26.2 ± 0.9 s−1. C, shown is deac-aminoATP cleavage and Pi release. The concentrations were as for panel B. The binding traces were repeated from panel B. Deac-aminoADP formation (circles) was measured using quench flow and HPLC. Pi release (dashed line) was measured in the presence of 10 μm rhodamine-PBP. The simulation in Fig. 5 was applied to fit the dataset. The simulated binding and cleavage are the dashed line in the main panel. The simulated Pi release is in the inset. This gave the observed first order rate constant for deac-aminoATP binding (equivalent to [PcrA] × k+1a) at 100 s−1 followed by a conformation change (equivalent to k+1b) at 29 s−1, hydrolytic cleavage at >100 s−1 and Pi release at >300 s−1.
Mentions: To test this hypothesis, a single turnover measurement, equivalent to Fig. 5, was done with PcrA helicase but using the fluorescent ATP analog, deac-aminoATP (16). Deac-aminoADP has a significantly tighter interaction with PcrA with a Kd of 1.7 μm (Fig. 10A), so almost all the diphosphate should remain bound during the single turnover experiment. After mixing excess PcrA·dT20 with deac-aminoATP, there was a biphasic increase in fluorescence (Fig. 10B). The initial increase in fluorescence, which accounted for 75% of the overall fluorescence change, had a rate constant of 130 s−1. This was followed by a slower change in fluorescence with a rate constant of 26 s−1. The first phase in fluorescence is presumably binding, whereas the second change could be due to a pre-cleavage conformation change. This was supported by measurements of hydrolysis by quench-flow and Pi release using a phosphate biosensor under the same conditions (Fig. 10B). To show the similarity in the mechanism between RecD2 and PcrA, the same model, used to fit data in Fig. 5, was applied to fit this dataset (Fig. 10C). This gave deac-aminoATP binding with a first order rate constant (equivalent to [PcrA] × k+1a; Fig. 1B) of 100 s−1 followed by a conformation change binding (equivalent to k+1b) of 29 s−1, hydrolytic cleavage at >100 s−1, and Pi release at >300 s−1.

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