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Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases.

ABSTRACT: Escherichia coli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3'-to-5' translocase, and RecD, a 5'-to-3' translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can "melt out" approximately 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg(2+)-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3'-(dT)(6) and 5'-(dT)(6) or 5'-(dT)(10)] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3'-(dT)(6) and 5'-(dT)(6) noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk(U)=774+/-16 bp s(-1)) and kinetic step size (m=3.3+/-1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k(C)=45+/-2 s(-1)). However, when the noncomplementary 5' ssDNA tail is extended to 10 nt [5'-(dT)(10) and 3'-(dT)(6)], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk(U)=538+/-24 bp s(-1)) is observed with a similar kinetic step size (m=3.9+/-0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5'-(dT)(6) and 3'-(dT)(6) tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k(C) initiation steps, with a similar kinetic step size (m=4.4+/-1.7 bp) and unwinding rate (mk(obs)=396+/-15 bp s(-1)). These results suggest that the additional kinetic steps with rate constant k(C) required for RecBCD to initiate unwinding of blunt-ended and twin (dT)(6)-tailed DNA reflect processes needed to engage the RecD motor with the 5' ssDNA.

SUBMITTER: Wu CG

PROVIDER: S-EPMC3174691 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases.

Wu Colin G CG Lohman Timothy M TM

Journal of molecular biology 20080716 2

Escherichia coli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3'-to-5' translocase, and RecD, a 5'-to-3' translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can "melt out" appr ...[more]

PMID: 18656489

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Project description:Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000?steps, we discover that transiently halting a single enzyme-DNA complex by depleting Mg(2+)-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1?second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1?minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity, but in fact reflects one microstate from the equilibrium ensemble that a single molecule manifests during an individual processive translocation event.

Dataset Information

Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases.

Publications

Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases.

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