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.

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ATPase reaction schemes.(A) Minimal mechanism for ATP hydrolysis by RecG (R) with DNA (D). Steps are numbered, such that step n has forward and reverse rate constants, k+n and k-n, respectively, and equilibrium constant, Kn. (B) Scheme for two-step binding of mantATP.
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pone-0038270-g002: ATPase reaction schemes.(A) Minimal mechanism for ATP hydrolysis by RecG (R) with DNA (D). Steps are numbered, such that step n has forward and reverse rate constants, k+n and k-n, respectively, and equilibrium constant, Kn. (B) Scheme for two-step binding of mantATP.

Mentions: Steady-state measurements were made to gain an overall assessment of the reaction and the affinities of various nucleotides before investigating the individual steps in the ATPase cycle. These steps are described by the basic scheme shown in Figure 2A. A two-strand DNA junction, which contains two 20-bp regions of complementary and non-complementary sequences (A40:B40 Figure 1 and Table S1), was used in all these measurements. This forms a Y-shape structure and RecG translocates one single-stranded arm, but then remains bound to the end of the junction still hydrolyzing ATP [10]. Use of this junction simplifies the system as the DNA substrate is unmodified by RecG action and more significantly, ssDNA is not produced.


ATPase cycle and DNA unwinding kinetics of RecG helicase.

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

ATPase reaction schemes.(A) Minimal mechanism for ATP hydrolysis by RecG (R) with DNA (D). Steps are numbered, such that step n has forward and reverse rate constants, k+n and k-n, respectively, and equilibrium constant, Kn. (B) Scheme for two-step binding of mantATP.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038270-g002: ATPase reaction schemes.(A) Minimal mechanism for ATP hydrolysis by RecG (R) with DNA (D). Steps are numbered, such that step n has forward and reverse rate constants, k+n and k-n, respectively, and equilibrium constant, Kn. (B) Scheme for two-step binding of mantATP.
Mentions: Steady-state measurements were made to gain an overall assessment of the reaction and the affinities of various nucleotides before investigating the individual steps in the ATPase cycle. These steps are described by the basic scheme shown in Figure 2A. A two-strand DNA junction, which contains two 20-bp regions of complementary and non-complementary sequences (A40:B40 Figure 1 and Table S1), was used in all these measurements. This forms a Y-shape structure and RecG translocates one single-stranded arm, but then remains bound to the end of the junction still hydrolyzing ATP [10]. Use of this junction simplifies the system as the DNA substrate is unmodified by RecG action and more significantly, ssDNA is not produced.

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