Limits...
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.

Show MeSH

Related in: MedlinePlus

Kinetic measurement of mantATP with excess of RecG and DNA: binding, hydrolysis and Pi release.The concentrations for all experiments were 0.5 µM mantATP, 2.5 µM RecG, 5 µM DNA (A40:B40) and 10 µM MDCC-PBP (for Pi measurement). All measurements were carried out at 20°C in the buffer described in Materials and Methods. (A) Time course of mant fluorescence (solid line), mantADP formation (circles) and Pi release (dashed line), measured as described in materials and methods. The inset shows the initial change in mant fluorescence. (B) Simulation of these time courses, based upon a global model for a single turnover of mantATP as described in the text. (C) The time course of mant fluorescence upon mixing 0.5 µM mantATP and 2.5 µM RecG with 5 µM DNA junction (A40:B40) after a first mixing of 5 µM RecG and 1 µM mantATP and aging for 1 s.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368886&req=5

pone-0038270-g004: Kinetic measurement of mantATP with excess of RecG and DNA: binding, hydrolysis and Pi release.The concentrations for all experiments were 0.5 µM mantATP, 2.5 µM RecG, 5 µM DNA (A40:B40) and 10 µM MDCC-PBP (for Pi measurement). All measurements were carried out at 20°C in the buffer described in Materials and Methods. (A) Time course of mant fluorescence (solid line), mantADP formation (circles) and Pi release (dashed line), measured as described in materials and methods. The inset shows the initial change in mant fluorescence. (B) Simulation of these time courses, based upon a global model for a single turnover of mantATP as described in the text. (C) The time course of mant fluorescence upon mixing 0.5 µM mantATP and 2.5 µM RecG with 5 µM DNA junction (A40:B40) after a first mixing of 5 µM RecG and 1 µM mantATP and aging for 1 s.

Mentions: Extra information relating to changes in a single ATPase cycle can be obtained by mixing excess RecG⋅DNA(A40:B40) with mantATP (Figure 4A). The traces showed an initial small increase in fluorescence (inset Figure 4A), which represents ∼20% of the overall change, followed by a slower, but larger increase. The first change in fluorescence is presumably due to binding. After fitting to a double exponential, the observed rate constant for the initial change was linearly dependent on RecG concentration over the small range possible (0.5 µM –2.5 µM), limited by protein precipitation at higher concentrations. Due to the low concentrations, a hyperbolic dependence would not be observed. The observed rate constant of the second increase in fluorescence, 0.22 (±0.05) s−1 (Figure 4A), was independent of RecG concentration. This second phase is likely to be due to the cleavage step, leading to the formation of bound mantADP. The fluorescence intensity does not change subsequently because almost all mantADP would remain bound at the concentrations used.


ATPase cycle and DNA unwinding kinetics of RecG helicase.

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

Kinetic measurement of mantATP with excess of RecG and DNA: binding, hydrolysis and Pi release.The concentrations for all experiments were 0.5 µM mantATP, 2.5 µM RecG, 5 µM DNA (A40:B40) and 10 µM MDCC-PBP (for Pi measurement). All measurements were carried out at 20°C in the buffer described in Materials and Methods. (A) Time course of mant fluorescence (solid line), mantADP formation (circles) and Pi release (dashed line), measured as described in materials and methods. The inset shows the initial change in mant fluorescence. (B) Simulation of these time courses, based upon a global model for a single turnover of mantATP as described in the text. (C) The time course of mant fluorescence upon mixing 0.5 µM mantATP and 2.5 µM RecG with 5 µM DNA junction (A40:B40) after a first mixing of 5 µM RecG and 1 µM mantATP and aging for 1 s.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368886&req=5

pone-0038270-g004: Kinetic measurement of mantATP with excess of RecG and DNA: binding, hydrolysis and Pi release.The concentrations for all experiments were 0.5 µM mantATP, 2.5 µM RecG, 5 µM DNA (A40:B40) and 10 µM MDCC-PBP (for Pi measurement). All measurements were carried out at 20°C in the buffer described in Materials and Methods. (A) Time course of mant fluorescence (solid line), mantADP formation (circles) and Pi release (dashed line), measured as described in materials and methods. The inset shows the initial change in mant fluorescence. (B) Simulation of these time courses, based upon a global model for a single turnover of mantATP as described in the text. (C) The time course of mant fluorescence upon mixing 0.5 µM mantATP and 2.5 µM RecG with 5 µM DNA junction (A40:B40) after a first mixing of 5 µM RecG and 1 µM mantATP and aging for 1 s.
Mentions: Extra information relating to changes in a single ATPase cycle can be obtained by mixing excess RecG⋅DNA(A40:B40) with mantATP (Figure 4A). The traces showed an initial small increase in fluorescence (inset Figure 4A), which represents ∼20% of the overall change, followed by a slower, but larger increase. The first change in fluorescence is presumably due to binding. After fitting to a double exponential, the observed rate constant for the initial change was linearly dependent on RecG concentration over the small range possible (0.5 µM –2.5 µM), limited by protein precipitation at higher concentrations. Due to the low concentrations, a hyperbolic dependence would not be observed. The observed rate constant of the second increase in fluorescence, 0.22 (±0.05) s−1 (Figure 4A), was independent of RecG concentration. This second phase is likely to be due to the cleavage step, leading to the formation of bound mantADP. The fluorescence intensity does not change subsequently because almost all mantADP would remain bound at the concentrations used.

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