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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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Mant fluorescent changes upon binding RecD2·dT20.A, shown is the time course of mant fluorescence upon mixing 2.5 μm mantATPγS and 12.5 μm RecD2·dT20. The inset shows the initial increase in fluorescence. The long time traces were fitted to double exponentials (Equation 2, dashed line) giving observed rate constants of 256 ± 8 and 27.4 ± 2.3 s−1. The single exponential fit is shown for comparison (dotted line). B, shown is the time course of mant fluorescence upon mixing 2.5 μm mantATP and 12.5 μm RecD2 with 15 μm dT20 after a first mixing of 25 μm RecD2 and 5 μm mantATP and aging for 0.1 s. The trace was fitted by a single exponential (Equation 1, dotted line) giving a rate constant of 48.2 ± 4.5 s−1. If the second mix was with buffer alone (no DNA), there was a small change in fluorescence, which increase linearly over a long time (not shown). This probably represents a basal level of activity.
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Figure 4: Mant fluorescent changes upon binding RecD2·dT20.A, shown is the time course of mant fluorescence upon mixing 2.5 μm mantATPγS and 12.5 μm RecD2·dT20. The inset shows the initial increase in fluorescence. The long time traces were fitted to double exponentials (Equation 2, dashed line) giving observed rate constants of 256 ± 8 and 27.4 ± 2.3 s−1. The single exponential fit is shown for comparison (dotted line). B, shown is the time course of mant fluorescence upon mixing 2.5 μm mantATP and 12.5 μm RecD2 with 15 μm dT20 after a first mixing of 25 μm RecD2 and 5 μm mantATP and aging for 0.1 s. The trace was fitted by a single exponential (Equation 1, dotted line) giving a rate constant of 48.2 ± 4.5 s−1. If the second mix was with buffer alone (no DNA), there was a small change in fluorescence, which increase linearly over a long time (not shown). This probably represents a basal level of activity.

Mentions: To probe the basis of the fluorescent changes, the experiments were repeated at a single concentration with the ATP analog, mantATPγS. This analog is hydrolyzed only very slowly by RecD2·dT20 (0.3 s−1), shown using the steady-state assay described above (Table 1). This was confirmed by HPLC analysis (data not shown). The traces of the single-turnover measurement with mantATPγS in Fig. 4A showed a biphasic increase in fluorescence very similar to that with mantATP. The observed rate constants were 256 s−1 for the first phase and 27.4 s−1 for the second. An equivalent single-turnover Pi measurement (as described below for mantATP) showed no Pi formation over the time period of the mant measurement, confirming that this nucleotide was essentially not hydrolyzed on this time scale. The two fluorescence phases observed with mantATPγS are very likely to be related to binding and a subsequent conformation change to the mantATPγS complex but cannot be due to cleavage.


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

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

Mant fluorescent changes upon binding RecD2·dT20.A, shown is the time course of mant fluorescence upon mixing 2.5 μm mantATPγS and 12.5 μm RecD2·dT20. The inset shows the initial increase in fluorescence. The long time traces were fitted to double exponentials (Equation 2, dashed line) giving observed rate constants of 256 ± 8 and 27.4 ± 2.3 s−1. The single exponential fit is shown for comparison (dotted line). B, shown is the time course of mant fluorescence upon mixing 2.5 μm mantATP and 12.5 μm RecD2 with 15 μm dT20 after a first mixing of 25 μm RecD2 and 5 μm mantATP and aging for 0.1 s. The trace was fitted by a single exponential (Equation 1, dotted line) giving a rate constant of 48.2 ± 4.5 s−1. If the second mix was with buffer alone (no DNA), there was a small change in fluorescence, which increase linearly over a long time (not shown). This probably represents a basal level of activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Mant fluorescent changes upon binding RecD2·dT20.A, shown is the time course of mant fluorescence upon mixing 2.5 μm mantATPγS and 12.5 μm RecD2·dT20. The inset shows the initial increase in fluorescence. The long time traces were fitted to double exponentials (Equation 2, dashed line) giving observed rate constants of 256 ± 8 and 27.4 ± 2.3 s−1. The single exponential fit is shown for comparison (dotted line). B, shown is the time course of mant fluorescence upon mixing 2.5 μm mantATP and 12.5 μm RecD2 with 15 μm dT20 after a first mixing of 25 μm RecD2 and 5 μm mantATP and aging for 0.1 s. The trace was fitted by a single exponential (Equation 1, dotted line) giving a rate constant of 48.2 ± 4.5 s−1. If the second mix was with buffer alone (no DNA), there was a small change in fluorescence, which increase linearly over a long time (not shown). This probably represents a basal level of activity.
Mentions: To probe the basis of the fluorescent changes, the experiments were repeated at a single concentration with the ATP analog, mantATPγS. This analog is hydrolyzed only very slowly by RecD2·dT20 (0.3 s−1), shown using the steady-state assay described above (Table 1). This was confirmed by HPLC analysis (data not shown). The traces of the single-turnover measurement with mantATPγS in Fig. 4A showed a biphasic increase in fluorescence very similar to that with mantATP. The observed rate constants were 256 s−1 for the first phase and 27.4 s−1 for the second. An equivalent single-turnover Pi measurement (as described below for mantATP) showed no Pi formation over the time period of the mant measurement, confirming that this nucleotide was essentially not hydrolyzed on this time scale. The two fluorescence phases observed with mantATPγS are very likely to be related to binding and a subsequent conformation change to the mantATPγS complex but cannot be due to cleavage.

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