Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation

Simon, Michael J.; Sokoloski, Joshua E.; Hao, Linxuan; Weiland, Elizabeth; Lohman, Timothy M.

doi:10.1016/j.jmb.2016.07.002

Cited by 13 publications

(34 citation statements)

References 63 publications

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“…However, a series of biochemical and biophysical studies support an alternative mechanism in which DNA melting of multiple base pairs occurs in an ATP‐independent reaction and that ATP hydrolysis is used primarily for directed ssDNA translocation of the enzyme. We first summarize the evidence that leads to this model.…”

Section: A Mechanical Model For Recbcd‐catalyzed Helicase Activitymentioning

confidence: 99%

“…That is, processive DNA unwinding should not occur without ssDNA translocation. Simon et al recently tested whether RecBCD could unwind duplex DNA in the absence of ssDNA translocation by its canonical RecB and RecD motors. DNA duplexes were designed with a unique initiation site for RecBCD, but contained so‐called “reversed polarity” (RP) linkages within each DNA strand just after the RecBCD binding site.…”

Section: Processive Duplex Dna Unwinding By Recbcd Can Be Uncoupled Fmentioning

confidence: 99%

“…These are positioned within each strand at the same position in the duplex DNA. Since SF1 ssDNA translocases are strictly directional, a 3′‐3′ linkage will stop the translocation of a canonical 3′ to 5′ translocase motor, and a 5′‐5′ linkage will stop the translocation of a canonical 5′ to 3′ translocase motor . Hence, the RecB and RecD canonical motors are unable to move past the point in the duplex DNA that contains the RP linkages.…”

Section: Processive Duplex Dna Unwinding By Recbcd Can Be Uncoupled Fmentioning

confidence: 99%

“…Surprisingly, RecBCD was able to processively unwind duplex DNA for at least 80 bp beyond the RP linkages with rates only half (≈400 bp/s) those during unwinding of natural DNA (≈800 bp/s). Although the canonical RecB and RecD motors are unable to bypass the RP linkages, another region of the RecBCD helicase must be capable of moving along and unwinding the duplex DNA in an ATP‐dependent process . Although RecBCD transiently unwinds the duplex DNA, once all of the ATP is hydrolyzed, the DNA substrate reanneals to its original state as long as the RecBCD enzyme remains bound to the DNA .…”

Section: Processive Duplex Dna Unwinding By Recbcd Can Be Uncoupled Fmentioning

confidence: 99%

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How Does a Helicase Unwind DNA? Insights from RecBCD Helicase

Lohman

Fazio

2018

BioEssays

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DNA helicases are a class of molecular motors that catalyze processive unwinding of double stranded DNA. In spite of much study, we know relatively little about the mechanisms by which these enzymes carry out the function for which they are named. Most current views are based on inferences from crystal structures. A prominent view is that the canonical ATPase motor exerts a force on the ssDNA resulting in "pulling" the duplex across a "pin" or "wedge" in the enzyme leading to a mechanical separation of the two DNA strands. In such models, DNA base pair separation is tightly coupled to ssDNA translocation of the motors. However, recent studies of the Escherichia coli RecBCD helicase suggest an alternative model in which DNA base pair melting and ssDNA translocation occur separately. In this view, the enzyme-DNA binding free energy is used to melt multiple DNA base pairs in an ATP-independent manner, followed by ATP-dependent translocation of the canonical motors along the newly formed ssDNA tracks. Repetition of these two steps results in processive DNA unwinding. We summarize recent evidence suggesting this mechanism for RecBCD helicase action.

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Section: A Mechanical Model For Recbcd‐catalyzed Helicase Activitymentioning

confidence: 99%

Section: Processive Duplex Dna Unwinding By Recbcd Can Be Uncoupled Fmentioning

confidence: 99%

Section: Processive Duplex Dna Unwinding By Recbcd Can Be Uncoupled Fmentioning

confidence: 99%

Section: Processive Duplex Dna Unwinding By Recbcd Can Be Uncoupled Fmentioning

confidence: 99%

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How Does a Helicase Unwind DNA? Insights from RecBCD Helicase

Lohman

Fazio

2018

BioEssays

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“…UvrD, PcrA, and Rep monomers [47–49]) and not all helicases require canonical protein translocation (e.g. RecBCD [50]), for helicases that translocate on ssDNA and unwind dsDNA, two limiting cases were proposed to provide a general framework for understanding the mechanism of strand separation [14]. Specifically, active helicases directly destabilize the downstream base-pair to catalyze the formation of ssDNA template prior to translocation while passive helicases do not.…”

Section: Active and Passive Nucleic Acid Duplex Unwindingmentioning

confidence: 99%

Conformational selection and induced fit as a useful framework for molecular motor mechanisms

Galburt

Tomko

2017

Biophysical Chemistry

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In this review, we explore mappings between mechanisms of ligand-binding, molecular motor translocation, and helicase unwinding of nucleic acids. The linkage between macromolecular binding and conformational change that is ubiquitous in biological molecules can be understood in the context of the of conformational selection and induced fit. The mechanism of biased motion exhibited by molecular motors is typically described as either a thermal ratchet or a power-stroke. Lastly, helicases are characterized by either active or passive unwinding mechanisms. We posit that both Brownian ratchet translocation and passive unwinding are examples of conformational selection and that both power-stroke translocation and active unwinding are examples of induced fit. In ligand-binding reactions, both conformational selection and induced fit may exist in parallel and the flux through either mechanistic path depends on ligand concentration. Given these mappings, we propose, in analogy with ligand-binding mechanisms, that motors may be able to function via parallel ratchet and stroke mechanisms and that helicases may be able to function via parallel active and passive mechanisms.

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How Chi Sequence Modifies RecBCD Single‐Stranded DNA Translocase Activity

Cho

Chung

2017

ChemPhysChem

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E. coli RecBCD initiates homologous repair as well as degrades foreign DNA. Recognition of chi sequence (5'-GCTGGTGG-3') switches RecBCD from a destructive, nucleolytic mode into a repair-active one that promotes RecA-mediated recombination. RecBCD includes a 3'-to-5' single-stranded DNA (ssDNA) translocase in RecB subunit, a 5'-to-3' translocase in RecD, and a secondary translocase activity associated with RecBC. To understand how chi specifically affects each translocase activity, we directly visualized individual RecBCD translocating along DNA substrates containing a ssDNA gap of different polarities, with or without chi. Disappearance of RecBCD from the ssDNA signals the loss of the ssDNA translocase activity. For substrates containing a ssDNA gap that RecBCD encounters in the 3'-to-5' polarity (3'-to-5' ssDNA), wild-type RecBCD disappears from the DNA substrates with similarly high percentage, either with chi or without. This suggests that (1) the 3'-to-5' translocase in RecB is unaffected by chi and (2) it is low in processivity. With substrates containing a ssDNA gap that RecBCD encounters in the 5'-to-3' polarity (5'-to-3' ssDNA), we found that the leaving percentage increases significantly with chi, implying inactivation of the 5'-to-3' translocase of RecD upon chi recognition. Surprisingly, the RecD defective mutant RecBCD showed only ≈50 % leaving on 5'-to-3' ssDNA, directly revealing the presence of RecBC secondary translocase and its activity is unaffected by chi. Multiple ssDNA translocases within the RecBCD complex both before and after chi ensures processive unwinding of DNA substrates required for efficient recombination events.

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Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation

Cited by 13 publications

References 63 publications

How Does a Helicase Unwind DNA? Insights from RecBCD Helicase

How Does a Helicase Unwind DNA? Insights from RecBCD Helicase

Conformational selection and induced fit as a useful framework for molecular motor mechanisms

How Chi Sequence Modifies RecBCD Single‐Stranded DNA Translocase Activity

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