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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Related in: MedlinePlus

DNA junctions and unwinding scheme.(A) Schematic representation of a model DNA substrate. The template strand forms the Ax series, where x is the number of nucleotides. The leading strand forms the Bx, if non-complementary, or the B′x series, if complementary. The lagging strand forms the Cx series. The strand complementary to the leading strand forms the Dx series. (B) Cartoon scheme showing unwinding of a complementary DNA junction by RecG. This example has a 5′-Cy3 label (white circle) on the template strand and a 3′-Dabcyl on the lagging strand (black circle).
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pone-0038270-g001: DNA junctions and unwinding scheme.(A) Schematic representation of a model DNA substrate. The template strand forms the Ax series, where x is the number of nucleotides. The leading strand forms the Bx, if non-complementary, or the B′x series, if complementary. The lagging strand forms the Cx series. The strand complementary to the leading strand forms the Dx series. (B) Cartoon scheme showing unwinding of a complementary DNA junction by RecG. This example has a 5′-Cy3 label (white circle) on the template strand and a 3′-Dabcyl on the lagging strand (black circle).

Mentions: A kinetic analysis of the ATPase cycle for Thermotoga maritima RecG is presented here to determine the main intermediates during the cycle and what biochemical steps may be coupled to translocation. The ATPase cycle was investigated by measuring rate constants for individual processes in the cycle. The complete cycle was measured using the analogue mantATP ((2′(3′)-O-(N-methylanthraniloyl)ATP), to provide a consistent view of the cycle, as some steps cannot be measured with the natural ATP substrate. However, because the nucleotide modification caused changes in kinetics for some parts of the ATPase cycle, several key steps were measured with the natural substrate, so that the ATPase mechanism with the ATP analogue could be related to that with unlabeled nucleotide. In particular mantATP is hydrolytically cleaved ∼10-fold slower than ATP and the release of mantADP is very slow. Despite these differences, the fluorescent ATP allowed definition of an ATPase mechanism and how this might be related to structural changes. Finally, DNA unwinding and corresponding ATPase measurements were used to relate the ATP usage with translocation for an expanded range of model substrates. This included homologous sequence throughout to mimic a stalled replication fork (Figure 1). These substrates contain four oligonucleotides, so that partial unwinding produces a chicken-foot structure and on complete unwinding two separate duplexes result.


ATPase cycle and DNA unwinding kinetics of RecG helicase.

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

DNA junctions and unwinding scheme.(A) Schematic representation of a model DNA substrate. The template strand forms the Ax series, where x is the number of nucleotides. The leading strand forms the Bx, if non-complementary, or the B′x series, if complementary. The lagging strand forms the Cx series. The strand complementary to the leading strand forms the Dx series. (B) Cartoon scheme showing unwinding of a complementary DNA junction by RecG. This example has a 5′-Cy3 label (white circle) on the template strand and a 3′-Dabcyl on the lagging strand (black circle).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038270-g001: DNA junctions and unwinding scheme.(A) Schematic representation of a model DNA substrate. The template strand forms the Ax series, where x is the number of nucleotides. The leading strand forms the Bx, if non-complementary, or the B′x series, if complementary. The lagging strand forms the Cx series. The strand complementary to the leading strand forms the Dx series. (B) Cartoon scheme showing unwinding of a complementary DNA junction by RecG. This example has a 5′-Cy3 label (white circle) on the template strand and a 3′-Dabcyl on the lagging strand (black circle).
Mentions: A kinetic analysis of the ATPase cycle for Thermotoga maritima RecG is presented here to determine the main intermediates during the cycle and what biochemical steps may be coupled to translocation. The ATPase cycle was investigated by measuring rate constants for individual processes in the cycle. The complete cycle was measured using the analogue mantATP ((2′(3′)-O-(N-methylanthraniloyl)ATP), to provide a consistent view of the cycle, as some steps cannot be measured with the natural ATP substrate. However, because the nucleotide modification caused changes in kinetics for some parts of the ATPase cycle, several key steps were measured with the natural substrate, so that the ATPase mechanism with the ATP analogue could be related to that with unlabeled nucleotide. In particular mantATP is hydrolytically cleaved ∼10-fold slower than ATP and the release of mantADP is very slow. Despite these differences, the fluorescent ATP allowed definition of an ATPase mechanism and how this might be related to structural changes. Finally, DNA unwinding and corresponding ATPase measurements were used to relate the ATP usage with translocation for an expanded range of model substrates. This included homologous sequence throughout to mimic a stalled replication fork (Figure 1). These substrates contain four oligonucleotides, so that partial unwinding produces a chicken-foot structure and on complete unwinding two separate duplexes result.

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