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ATPase cycle and DNA unwinding kinetics of RecG helicase.

Toseland CP, Powell B, Webb MR - PLoS ONE (2012)

Bottom Line: The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle.Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism.Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states.

View Article: PubMed Central - PubMed

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

ABSTRACT
The superfamily 2 bacterial helicase, RecG, is a monomeric enzyme with a role in DNA repair by reversing stalled replication forks. The helicase must act specifically and rapidly to prevent replication fork collapse. We have shown that RecG binds tightly and rapidly to four-strand oligonucleotide junctions, which mimic a stalled replication fork. The helicase unwinds such DNA junctions with a step-size of approximately four bases per ATP hydrolyzed. To gain an insight into this mechanism, we used fluorescent stopped-flow and quenched-flow to measure individual steps within the ATPase cycle of RecG, when bound to a DNA junction. The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle. Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism. The data show that the chemical step of hydrolysis is the rate limiting step in the cycle and that this step is greatly accelerated by bound DNA. The ADP release rate is similar to the cleavage rate, so that bound ATP and ADP would be the main states during the ATP cycle. Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states.

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ATPase cycle measurements with unmodified nucleotides.(A) Phosphate measurements at different ATP concentrations. 0.5 µM RecG, premixed with 2.5 µM DNA (A40:B40), was then mixed in the stopped-flow apparatus with ATP at the micromolar concentrations shown, in the presence of 10 µM MDCC-PBP. (B) Best fit time courses of the Pi release, based upon a model of the ATPase cycle, as described in the text. Curves were obtained by glogal fitting the curves at different concentrations, using Global kinetic Explorer [40]. Note that in comparison, the experimental time courses show a small transient of Pi at times <100 ms. This is likely to be due to free Pi and this behavior, overlaying a lag, has been observed previously in multi-turnover measurements [13]. (C) ADP Release kinetics. 10 µM RecG⋅DNA (A40∶B40) was pre-bound with 10 µM ADP before mixing with an excess of mantADP (20 µM): these concentrations are final in the mixing chamber. The increase in fluorescence was fitted to single exponential giving the dissociation rate constant of 11.4 (±2.2) s−1. Rate constants were independent of mantADP concentration in the range of 20–80 µM.
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pone-0038270-g006: ATPase cycle measurements with unmodified nucleotides.(A) Phosphate measurements at different ATP concentrations. 0.5 µM RecG, premixed with 2.5 µM DNA (A40:B40), was then mixed in the stopped-flow apparatus with ATP at the micromolar concentrations shown, in the presence of 10 µM MDCC-PBP. (B) Best fit time courses of the Pi release, based upon a model of the ATPase cycle, as described in the text. Curves were obtained by glogal fitting the curves at different concentrations, using Global kinetic Explorer [40]. Note that in comparison, the experimental time courses show a small transient of Pi at times <100 ms. This is likely to be due to free Pi and this behavior, overlaying a lag, has been observed previously in multi-turnover measurements [13]. (C) ADP Release kinetics. 10 µM RecG⋅DNA (A40∶B40) was pre-bound with 10 µM ADP before mixing with an excess of mantADP (20 µM): these concentrations are final in the mixing chamber. The increase in fluorescence was fitted to single exponential giving the dissociation rate constant of 11.4 (±2.2) s−1. Rate constants were independent of mantADP concentration in the range of 20–80 µM.

Mentions: The rate of Pi release was measured after mixing RecG⋅DNA with excess ATP, using the phosphate biosensor, in the stopped-flow apparatus (Figure 6A). The traces showed a slight lag, followed by a fairly linear rate of Pi release. Both phases are dependent on ATP concentration. The lag decreased, while the rate of the linear phase increased with greater concentrations of ATP. The lag is due to the steps prior to Pi release, namely ATP binding and cleavage. These traces represented multiple ATP turnovers and showed no burst of Pi release during the first turnover: therefore ADP release is not rate limiting in the cycle. Such a burst phase would result from the relatively fast Pi release of the initial cycle before the Pi release of subsequent cycles was limited by slow ADP release.


ATPase cycle and DNA unwinding kinetics of RecG helicase.

Toseland CP, Powell B, Webb MR - PLoS ONE (2012)

ATPase cycle measurements with unmodified nucleotides.(A) Phosphate measurements at different ATP concentrations. 0.5 µM RecG, premixed with 2.5 µM DNA (A40:B40), was then mixed in the stopped-flow apparatus with ATP at the micromolar concentrations shown, in the presence of 10 µM MDCC-PBP. (B) Best fit time courses of the Pi release, based upon a model of the ATPase cycle, as described in the text. Curves were obtained by glogal fitting the curves at different concentrations, using Global kinetic Explorer [40]. Note that in comparison, the experimental time courses show a small transient of Pi at times <100 ms. This is likely to be due to free Pi and this behavior, overlaying a lag, has been observed previously in multi-turnover measurements [13]. (C) ADP Release kinetics. 10 µM RecG⋅DNA (A40∶B40) was pre-bound with 10 µM ADP before mixing with an excess of mantADP (20 µM): these concentrations are final in the mixing chamber. The increase in fluorescence was fitted to single exponential giving the dissociation rate constant of 11.4 (±2.2) s−1. Rate constants were independent of mantADP concentration in the range of 20–80 µM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368886&req=5

pone-0038270-g006: ATPase cycle measurements with unmodified nucleotides.(A) Phosphate measurements at different ATP concentrations. 0.5 µM RecG, premixed with 2.5 µM DNA (A40:B40), was then mixed in the stopped-flow apparatus with ATP at the micromolar concentrations shown, in the presence of 10 µM MDCC-PBP. (B) Best fit time courses of the Pi release, based upon a model of the ATPase cycle, as described in the text. Curves were obtained by glogal fitting the curves at different concentrations, using Global kinetic Explorer [40]. Note that in comparison, the experimental time courses show a small transient of Pi at times <100 ms. This is likely to be due to free Pi and this behavior, overlaying a lag, has been observed previously in multi-turnover measurements [13]. (C) ADP Release kinetics. 10 µM RecG⋅DNA (A40∶B40) was pre-bound with 10 µM ADP before mixing with an excess of mantADP (20 µM): these concentrations are final in the mixing chamber. The increase in fluorescence was fitted to single exponential giving the dissociation rate constant of 11.4 (±2.2) s−1. Rate constants were independent of mantADP concentration in the range of 20–80 µM.
Mentions: The rate of Pi release was measured after mixing RecG⋅DNA with excess ATP, using the phosphate biosensor, in the stopped-flow apparatus (Figure 6A). The traces showed a slight lag, followed by a fairly linear rate of Pi release. Both phases are dependent on ATP concentration. The lag decreased, while the rate of the linear phase increased with greater concentrations of ATP. The lag is due to the steps prior to Pi release, namely ATP binding and cleavage. These traces represented multiple ATP turnovers and showed no burst of Pi release during the first turnover: therefore ADP release is not rate limiting in the cycle. Such a burst phase would result from the relatively fast Pi release of the initial cycle before the Pi release of subsequent cycles was limited by slow ADP release.

Bottom Line: The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle.Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism.Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states.

View Article: PubMed Central - PubMed

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

ABSTRACT
The superfamily 2 bacterial helicase, RecG, is a monomeric enzyme with a role in DNA repair by reversing stalled replication forks. The helicase must act specifically and rapidly to prevent replication fork collapse. We have shown that RecG binds tightly and rapidly to four-strand oligonucleotide junctions, which mimic a stalled replication fork. The helicase unwinds such DNA junctions with a step-size of approximately four bases per ATP hydrolyzed. To gain an insight into this mechanism, we used fluorescent stopped-flow and quenched-flow to measure individual steps within the ATPase cycle of RecG, when bound to a DNA junction. The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle. Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism. The data show that the chemical step of hydrolysis is the rate limiting step in the cycle and that this step is greatly accelerated by bound DNA. The ADP release rate is similar to the cleavage rate, so that bound ATP and ADP would be the main states during the ATP cycle. Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states.

Show MeSH
Related in: MedlinePlus