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

Unwinding 4-strand complementary junctions.(A) Fluorescence changes during unwinding under single turnover conditions junctions (A:B′:C:D) of varying arm lengths (shown in b.p.), labeled with Cy3 and Dabcyl on the template strand duplex. Final concentrations are 30 nM DNA junction, 60 nM RecG and 200 µM ATP. The reaction was initiated by rapid mixing with ATP. (B) Fluorescence changes during unwinding under single turnover conditions of 4-strand complementary junctions (A∶B′∶C∶D), of varying arm lengths (shown in b.p.), labeled with Cy3 and Dabcyl on the leading strand duplex. Final concentrations DNA junction 30 nM, RecG 60 nM and ATP 200 µM. The reaction was initiated by rapid mixing with ATP. Fluorescence levels are expressed relative to the starting value. (C) Change in lag durations with arm length of complementary junctions.: 4-strand with lagging strand labeled (triangles), 3-strand with lagging strand labeled (circles) and 4-strand with leading strand labeled (squares). The length of lags were obtained by the intercept of best fits to the lag phase and the tangent to the rise phase, Lines are linear fits to the data points, and the reciprocal gives rates of unwinding 26.9 (±1.6) bp s−1 for the lagging strand and 26.9 (±1.6) bp s−1 for the leading strand.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368886&req=5

pone-0038270-g007: Unwinding 4-strand complementary junctions.(A) Fluorescence changes during unwinding under single turnover conditions junctions (A:B′:C:D) of varying arm lengths (shown in b.p.), labeled with Cy3 and Dabcyl on the template strand duplex. Final concentrations are 30 nM DNA junction, 60 nM RecG and 200 µM ATP. The reaction was initiated by rapid mixing with ATP. (B) Fluorescence changes during unwinding under single turnover conditions of 4-strand complementary junctions (A∶B′∶C∶D), of varying arm lengths (shown in b.p.), labeled with Cy3 and Dabcyl on the leading strand duplex. Final concentrations DNA junction 30 nM, RecG 60 nM and ATP 200 µM. The reaction was initiated by rapid mixing with ATP. Fluorescence levels are expressed relative to the starting value. (C) Change in lag durations with arm length of complementary junctions.: 4-strand with lagging strand labeled (triangles), 3-strand with lagging strand labeled (circles) and 4-strand with leading strand labeled (squares). The length of lags were obtained by the intercept of best fits to the lag phase and the tangent to the rise phase, Lines are linear fits to the data points, and the reciprocal gives rates of unwinding 26.9 (±1.6) bp s−1 for the lagging strand and 26.9 (±1.6) bp s−1 for the leading strand.

Mentions: After measuring the ATPase kinetics and determining how they relate to interactions with DNA, it is important to correlate helicase activity with detailed enzymatic activity. Previously, unwinding measurements were reported with non-complementary three-way junctions [10]. RecG activity requires the substrate DNA to be a three-way junction, but the junction arms can be made up of completely duplex DNA or one arm, or both arms can be single strand, as used above. This diversity of DNA structures mimics the potential DNA substrates in the cell, depending on the precise extent of strand replication prior to the premature termination. It was therefore of interest to measure RecG activity on four-strand homologous junctions (Figure 7). The final products of unwinding are fully complementary, double strands. The kinetics of unwinding such DNA were measured with a Cy3/Dabcyl fluorophore/quencher pair at the distal end of the lagging strand, as done previously [10], [15]. In such substrates, the Dabcyl interacts with the Cy3 and quenches its fluorescence. However, on completion of unwinding, the two labels separate and the fluorescence increases (Figure 1B).


ATPase cycle and DNA unwinding kinetics of RecG helicase.

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

Unwinding 4-strand complementary junctions.(A) Fluorescence changes during unwinding under single turnover conditions junctions (A:B′:C:D) of varying arm lengths (shown in b.p.), labeled with Cy3 and Dabcyl on the template strand duplex. Final concentrations are 30 nM DNA junction, 60 nM RecG and 200 µM ATP. The reaction was initiated by rapid mixing with ATP. (B) Fluorescence changes during unwinding under single turnover conditions of 4-strand complementary junctions (A∶B′∶C∶D), of varying arm lengths (shown in b.p.), labeled with Cy3 and Dabcyl on the leading strand duplex. Final concentrations DNA junction 30 nM, RecG 60 nM and ATP 200 µM. The reaction was initiated by rapid mixing with ATP. Fluorescence levels are expressed relative to the starting value. (C) Change in lag durations with arm length of complementary junctions.: 4-strand with lagging strand labeled (triangles), 3-strand with lagging strand labeled (circles) and 4-strand with leading strand labeled (squares). The length of lags were obtained by the intercept of best fits to the lag phase and the tangent to the rise phase, Lines are linear fits to the data points, and the reciprocal gives rates of unwinding 26.9 (±1.6) bp s−1 for the lagging strand and 26.9 (±1.6) bp s−1 for the leading strand.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038270-g007: Unwinding 4-strand complementary junctions.(A) Fluorescence changes during unwinding under single turnover conditions junctions (A:B′:C:D) of varying arm lengths (shown in b.p.), labeled with Cy3 and Dabcyl on the template strand duplex. Final concentrations are 30 nM DNA junction, 60 nM RecG and 200 µM ATP. The reaction was initiated by rapid mixing with ATP. (B) Fluorescence changes during unwinding under single turnover conditions of 4-strand complementary junctions (A∶B′∶C∶D), of varying arm lengths (shown in b.p.), labeled with Cy3 and Dabcyl on the leading strand duplex. Final concentrations DNA junction 30 nM, RecG 60 nM and ATP 200 µM. The reaction was initiated by rapid mixing with ATP. Fluorescence levels are expressed relative to the starting value. (C) Change in lag durations with arm length of complementary junctions.: 4-strand with lagging strand labeled (triangles), 3-strand with lagging strand labeled (circles) and 4-strand with leading strand labeled (squares). The length of lags were obtained by the intercept of best fits to the lag phase and the tangent to the rise phase, Lines are linear fits to the data points, and the reciprocal gives rates of unwinding 26.9 (±1.6) bp s−1 for the lagging strand and 26.9 (±1.6) bp s−1 for the leading strand.
Mentions: After measuring the ATPase kinetics and determining how they relate to interactions with DNA, it is important to correlate helicase activity with detailed enzymatic activity. Previously, unwinding measurements were reported with non-complementary three-way junctions [10]. RecG activity requires the substrate DNA to be a three-way junction, but the junction arms can be made up of completely duplex DNA or one arm, or both arms can be single strand, as used above. This diversity of DNA structures mimics the potential DNA substrates in the cell, depending on the precise extent of strand replication prior to the premature termination. It was therefore of interest to measure RecG activity on four-strand homologous junctions (Figure 7). The final products of unwinding are fully complementary, double strands. The kinetics of unwinding such DNA were measured with a Cy3/Dabcyl fluorophore/quencher pair at the distal end of the lagging strand, as done previously [10], [15]. In such substrates, the Dabcyl interacts with the Cy3 and quenches its fluorescence. However, on completion of unwinding, the two labels separate and the fluorescence increases (Figure 1B).

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