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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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MantADP binding kinetics to RecG⋅DNA.MantADP at various concentrations was mixed in the stopped flow apparatus with 0.5 µM RecG and 2.5 µM DNA (A40:B40) under the conditions of Figure 4. Traces (not shown) were fitted by single exponentials. The rate constants are shown as a function of concentration and the best linear fit, gives a slope of 1.1 (±0.4) µM−1s−1 and an intercept of <1 s−1.
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pone-0038270-g005: MantADP binding kinetics to RecG⋅DNA.MantADP at various concentrations was mixed in the stopped flow apparatus with 0.5 µM RecG and 2.5 µM DNA (A40:B40) under the conditions of Figure 4. Traces (not shown) were fitted by single exponentials. The rate constants are shown as a function of concentration and the best linear fit, gives a slope of 1.1 (±0.4) µM−1s−1 and an intercept of <1 s−1.

Mentions: MantADP binding to RecG⋅DNA complex was measured under pseudo-first order conditions by stopped-flow fluorescence. The traces showed a single exponential increase. Figure 5 shows a linear dependence between observed rate constants and mantADP concentration, giving a second order rate constant (k−4 in the scheme of Figure 2A) of 1.1 (±0.4) µM−1 s−1 and a dissociation rate constant (k+4) of <1 s−1 from the intercept (Table 2).


ATPase cycle and DNA unwinding kinetics of RecG helicase.

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

MantADP binding kinetics to RecG⋅DNA.MantADP at various concentrations was mixed in the stopped flow apparatus with 0.5 µM RecG and 2.5 µM DNA (A40:B40) under the conditions of Figure 4. Traces (not shown) were fitted by single exponentials. The rate constants are shown as a function of concentration and the best linear fit, gives a slope of 1.1 (±0.4) µM−1s−1 and an intercept of <1 s−1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038270-g005: MantADP binding kinetics to RecG⋅DNA.MantADP at various concentrations was mixed in the stopped flow apparatus with 0.5 µM RecG and 2.5 µM DNA (A40:B40) under the conditions of Figure 4. Traces (not shown) were fitted by single exponentials. The rate constants are shown as a function of concentration and the best linear fit, gives a slope of 1.1 (±0.4) µM−1s−1 and an intercept of <1 s−1.
Mentions: MantADP binding to RecG⋅DNA complex was measured under pseudo-first order conditions by stopped-flow fluorescence. The traces showed a single exponential increase. Figure 5 shows a linear dependence between observed rate constants and mantADP concentration, giving a second order rate constant (k−4 in the scheme of Figure 2A) of 1.1 (±0.4) µM−1 s−1 and a dissociation rate constant (k+4) of <1 s−1 from the intercept (Table 2).

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