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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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ATP hydrolysis during unwinding 4-strand complementary junctions.Junctions of varying arm lengths (shown in b.p.) were used to measure the rate of ATP hydrolysis during unwinding. RecG was pre-incubated with junction and the reactions were initiated by rapid mixing with ATP and heparin. Final concentrations are 200 nM DNA junction (A∶B′∶C∶D), 10 nM RecG, 200 µM ATP, 1 mg ml−1 (55.5 µM) heparin and 5 µM MDCC-PBP. The reactions were performed in the presence of a Pi mop, described in materials and methods. Inset: Effect of heparin on ATPase activity of RecG during unwinding. The reactions were initiated by rapidly mixing the pre-incubated RecG.DNA (A40:B′40:C19) with ATP with or without heparin. In another measurement (“control”), RecG was pre-incubated with heparin and then rapidly mixed with DNA junction (A40:B′40:C19) plus ATP. Final concentrations are 200 nM DNA junction, 10 nM RecG, 200 µM ATP, 2 mg ml−1 (111 µM) heparin and 5 µM MDCC-PBP with Pi mop.
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pone-0038270-g008: ATP hydrolysis during unwinding 4-strand complementary junctions.Junctions of varying arm lengths (shown in b.p.) were used to measure the rate of ATP hydrolysis during unwinding. RecG was pre-incubated with junction and the reactions were initiated by rapid mixing with ATP and heparin. Final concentrations are 200 nM DNA junction (A∶B′∶C∶D), 10 nM RecG, 200 µM ATP, 1 mg ml−1 (55.5 µM) heparin and 5 µM MDCC-PBP. The reactions were performed in the presence of a Pi mop, described in materials and methods. Inset: Effect of heparin on ATPase activity of RecG during unwinding. The reactions were initiated by rapidly mixing the pre-incubated RecG.DNA (A40:B′40:C19) with ATP with or without heparin. In another measurement (“control”), RecG was pre-incubated with heparin and then rapidly mixed with DNA junction (A40:B′40:C19) plus ATP. Final concentrations are 200 nM DNA junction, 10 nM RecG, 200 µM ATP, 2 mg ml−1 (111 µM) heparin and 5 µM MDCC-PBP with Pi mop.

Mentions: The rate of ATP hydrolysis and total ATP usage during unwinding were measured using the phosphate biosensor (Figure 8), following rapid mixing of ATP with the RecG⋅DNA complex. A measurement of Pi production during unwinding showed a break point in the trace when heparin was used as a trap (Figure 8 inset). Heparin is a potent inhibitor of many helicases, mimicking the DNA substrate and binding tightly to the enzyme, once the latter dissociates from the DNA, and so prevents rebinding of the helicase to fresh DNA substrates. The initial, rapid phase, which represents unwinding of the first DNA junction, was unaffected by the presence of heparin. However, the subsequent change in rate is slow, suggesting that the interaction with heparin is slow, possibly due to slow release of RecG from the end of the DNA, or due to inherently slow interaction of RecG with heparin. RecG will continue to hydrolyze ATP while bound to the DNA substrate. Therefore, this does not produce an abrupt change in rate at the end of unwinding the first DNA substrate.


ATPase cycle and DNA unwinding kinetics of RecG helicase.

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

ATP hydrolysis during unwinding 4-strand complementary junctions.Junctions of varying arm lengths (shown in b.p.) were used to measure the rate of ATP hydrolysis during unwinding. RecG was pre-incubated with junction and the reactions were initiated by rapid mixing with ATP and heparin. Final concentrations are 200 nM DNA junction (A∶B′∶C∶D), 10 nM RecG, 200 µM ATP, 1 mg ml−1 (55.5 µM) heparin and 5 µM MDCC-PBP. The reactions were performed in the presence of a Pi mop, described in materials and methods. Inset: Effect of heparin on ATPase activity of RecG during unwinding. The reactions were initiated by rapidly mixing the pre-incubated RecG.DNA (A40:B′40:C19) with ATP with or without heparin. In another measurement (“control”), RecG was pre-incubated with heparin and then rapidly mixed with DNA junction (A40:B′40:C19) plus ATP. Final concentrations are 200 nM DNA junction, 10 nM RecG, 200 µM ATP, 2 mg ml−1 (111 µM) heparin and 5 µM MDCC-PBP with Pi mop.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368886&req=5

pone-0038270-g008: ATP hydrolysis during unwinding 4-strand complementary junctions.Junctions of varying arm lengths (shown in b.p.) were used to measure the rate of ATP hydrolysis during unwinding. RecG was pre-incubated with junction and the reactions were initiated by rapid mixing with ATP and heparin. Final concentrations are 200 nM DNA junction (A∶B′∶C∶D), 10 nM RecG, 200 µM ATP, 1 mg ml−1 (55.5 µM) heparin and 5 µM MDCC-PBP. The reactions were performed in the presence of a Pi mop, described in materials and methods. Inset: Effect of heparin on ATPase activity of RecG during unwinding. The reactions were initiated by rapidly mixing the pre-incubated RecG.DNA (A40:B′40:C19) with ATP with or without heparin. In another measurement (“control”), RecG was pre-incubated with heparin and then rapidly mixed with DNA junction (A40:B′40:C19) plus ATP. Final concentrations are 200 nM DNA junction, 10 nM RecG, 200 µM ATP, 2 mg ml−1 (111 µM) heparin and 5 µM MDCC-PBP with Pi mop.
Mentions: The rate of ATP hydrolysis and total ATP usage during unwinding were measured using the phosphate biosensor (Figure 8), following rapid mixing of ATP with the RecG⋅DNA complex. A measurement of Pi production during unwinding showed a break point in the trace when heparin was used as a trap (Figure 8 inset). Heparin is a potent inhibitor of many helicases, mimicking the DNA substrate and binding tightly to the enzyme, once the latter dissociates from the DNA, and so prevents rebinding of the helicase to fresh DNA substrates. The initial, rapid phase, which represents unwinding of the first DNA junction, was unaffected by the presence of heparin. However, the subsequent change in rate is slow, suggesting that the interaction with heparin is slow, possibly due to slow release of RecG from the end of the DNA, or due to inherently slow interaction of RecG with heparin. RecG will continue to hydrolyze ATP while bound to the DNA substrate. Therefore, this does not produce an abrupt change in rate at the end of unwinding the first DNA substrate.

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