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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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MantATP binding to RecG⋅DNA.MantATP at the micromolar concentrations shown was mixed in the stopped flow apparatus with 0.5 µM RecG and 2.5 µM DNA Junction (A40:B40) at 20°C in the buffer described in materials and methods. Individual traces (offset from each other) were fitted to single exponentials and the dependence of the rate constants on concentration was then fitted by a hyperbola. The points shown are averages of at least 3 measurements. The fit gives 1/K1a as 24 (±4) µM and k+1b + k−1b + k+2 as 26.1 (±1.8) s−1 and the intercept was <1 s–1 (Scheme in Figure 2B).
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pone-0038270-g003: MantATP binding to RecG⋅DNA.MantATP at the micromolar concentrations shown was mixed in the stopped flow apparatus with 0.5 µM RecG and 2.5 µM DNA Junction (A40:B40) at 20°C in the buffer described in materials and methods. Individual traces (offset from each other) were fitted to single exponentials and the dependence of the rate constants on concentration was then fitted by a hyperbola. The points shown are averages of at least 3 measurements. The fit gives 1/K1a as 24 (±4) µM and k+1b + k−1b + k+2 as 26.1 (±1.8) s−1 and the intercept was <1 s–1 (Scheme in Figure 2B).

Mentions: MantATP binding to RecG⋅DNA(A40:B40) was measured under pseudo-first-order conditions, that is mantATP in large excess over the protein. Using the stopped-flow apparatus, several concentrations of mantATP were rapidly mixed with RecG⋅DNA and fluorescence followed with time. Each trace was fitted by a single exponential (Figure 3A) and the concentration dependence of the observed rate constants were fit well by a hyperbola (Figure 3B). This suggests that the binding occurs in two steps with the first step being rapid and the second step having the predominant fluorescence change, as shown in the scheme (Figure 2B). As mantATP is also hydrolyzed, the rate of the forward reaction needs to be included, in this case limited by the cleavage step 2, as described later. The fit gives 1/K1a as 24 (±4) µM, k+1b+ k−1b + k+2 as 26.1 (±1.8) s−1 and the intercept gives k−1b + k+2 as <1 s−1. Given that k+2 is 0.2 s–1 (see below), k−1b is <0.8 s−1. Potentially, an alternative explanation for such a hyperbolic relationship could be preferential binding of either the 2′- or 3′-isomer of mantATP and then isomerization of the non-favored isomer. However, the observed rates in these measurements were several orders of magnitude faster than the likely isomerization rate [12]. In order to assess whether the two isomers of mantATP and their interconversion are factors in any fluorescence traces for mantATP, an equivalent measurement was made using 3′-mant-2′-deoxyATP. The data (not shown) was similar to the mixed isomers. Rate constants for individual steps in the mantATPase mechanism are summarized in Table 2.


ATPase cycle and DNA unwinding kinetics of RecG helicase.

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

MantATP binding to RecG⋅DNA.MantATP at the micromolar concentrations shown was mixed in the stopped flow apparatus with 0.5 µM RecG and 2.5 µM DNA Junction (A40:B40) at 20°C in the buffer described in materials and methods. Individual traces (offset from each other) were fitted to single exponentials and the dependence of the rate constants on concentration was then fitted by a hyperbola. The points shown are averages of at least 3 measurements. The fit gives 1/K1a as 24 (±4) µM and k+1b + k−1b + k+2 as 26.1 (±1.8) s−1 and the intercept was <1 s–1 (Scheme in Figure 2B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368886&req=5

pone-0038270-g003: MantATP binding to RecG⋅DNA.MantATP at the micromolar concentrations shown was mixed in the stopped flow apparatus with 0.5 µM RecG and 2.5 µM DNA Junction (A40:B40) at 20°C in the buffer described in materials and methods. Individual traces (offset from each other) were fitted to single exponentials and the dependence of the rate constants on concentration was then fitted by a hyperbola. The points shown are averages of at least 3 measurements. The fit gives 1/K1a as 24 (±4) µM and k+1b + k−1b + k+2 as 26.1 (±1.8) s−1 and the intercept was <1 s–1 (Scheme in Figure 2B).
Mentions: MantATP binding to RecG⋅DNA(A40:B40) was measured under pseudo-first-order conditions, that is mantATP in large excess over the protein. Using the stopped-flow apparatus, several concentrations of mantATP were rapidly mixed with RecG⋅DNA and fluorescence followed with time. Each trace was fitted by a single exponential (Figure 3A) and the concentration dependence of the observed rate constants were fit well by a hyperbola (Figure 3B). This suggests that the binding occurs in two steps with the first step being rapid and the second step having the predominant fluorescence change, as shown in the scheme (Figure 2B). As mantATP is also hydrolyzed, the rate of the forward reaction needs to be included, in this case limited by the cleavage step 2, as described later. The fit gives 1/K1a as 24 (±4) µM, k+1b+ k−1b + k+2 as 26.1 (±1.8) s−1 and the intercept gives k−1b + k+2 as <1 s−1. Given that k+2 is 0.2 s–1 (see below), k−1b is <0.8 s−1. Potentially, an alternative explanation for such a hyperbolic relationship could be preferential binding of either the 2′- or 3′-isomer of mantATP and then isomerization of the non-favored isomer. However, the observed rates in these measurements were several orders of magnitude faster than the likely isomerization rate [12]. In order to assess whether the two isomers of mantATP and their interconversion are factors in any fluorescence traces for mantATP, an equivalent measurement was made using 3′-mant-2′-deoxyATP. The data (not shown) was similar to the mixed isomers. Rate constants for individual steps in the mantATPase mechanism are summarized in 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
Related in: MedlinePlus