Escherichia coli RecBC helicase has two translocase activities controlled by a single ATPase motor

Wu, Colin G.; Bradford, Christina; Lohman, Timothy M.

doi:10.1038/nsmb.1901

Cited by 49 publications

(136 citation statements)

References 36 publications

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“…Difficulty in determining a kinetic stepsize for 5′-3′ translocation by RecBC was attributed to the possible presence of non-uniform processes (pausing, backward motion, etc) that may be occurring during RecBC translocation in this direction. An additional difference between these two translocase activities is that 3′-5′ translocation is blocked by reverse polarity linkages in the DNA, but 5′-3′ translocation is not (23). Despite these differences, ATPase is tightly coupled to translocation for both translocase activities of the RecB motor (29).…”

Section: Sf1 Family Helicasesmentioning

confidence: 99%

“…Isolated monomers of RecB have been shown to translocate from 3′ to 5′ along ssDNA with a macroscopic rate of (860 ± 53) nt/s and a kinetic step-size of (4.8 ± 0.3) nucleotides at saturating ATP concentrations (23). Interestingly, the RecBC translocase, which contains only the single RecB motor, is also capable of 5′-3′ translocation along ssDNA (23).…”

Section: Sf1 Family Helicasesmentioning

confidence: 99%

“…Interestingly, the RecBC translocase, which contains only the single RecB motor, is also capable of 5′-3′ translocation along ssDNA (23). At saturating ATP concentrations RecBC exhibits 3′-5′ translocation with a macroscopic rate and kinetic step-size of (920 ± 33) nt/s and (3.5 ± 0.2) nt, respectively, and 5′-3′ translocation with a macroscopic rate of (1060 ± 30) nt/s; when both translocase activities occur simultaneously the macroscopic rate of translocation is (671 ± 47) nt/s.…”

Section: Sf1 Family Helicasesmentioning

confidence: 99%

“…Several of the most commonly used methods of monitoring the translocation of a motor protein are: a) measuring the arrival of the protein at a particular point along the DNA (19)(20)(21)(22)(23)(24); b) detecting the displacement of obstacles from DNA by the translocating motor (25)(26)(27); c) DNA-dependent ATPase activity of the motor (18,22,(27)(28)(29)(30)(31); d) translocation-induced changes in the structure/ topology of the DNA (32,33). While each approach has advantages and disadvantages, addressing the key questions typically involves a combined approach of several of these techniques.…”

Section: Experimental Approaches and Mechanistic Modelsmentioning

confidence: 99%

“…For example, many motors are believed to form DNA loops during translocation (22)(23)(24)(25)(26). By considering motor efficiency, the distinction between kinetic and physical step-sizes of the translocation mechanism can be elucidated.…”

Section: Introductionmentioning

confidence: 99%

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All motors have to decide is what to do with the DNA that is given them

Briggs

Fischer

2014

Biomolecular Concepts

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DNA translocases are a diverse group of molecular motors responsible for a wide variety of cellular functions. The goal of this review is to identify common aspects in the mechanisms for how these enzymes couple the binding and hydrolysis of ATP to their movement along DNA. Not surprisingly, the shared structural components contained within the catalytic domains of several of these motors appear to give rise to common aspects of DNA translocation. Perhaps more interesting, however, are the differences between the families of translocases and the potential associated implications both for the functions of the members of these families and for the evolution of these families. However, as there are few translocases for which complete characterizations of the mechanisms of DNA binding, DNA translocation, and DNA-stimulated ATPase have been completed, it is difficult to form many inferences. We therefore hope that this review motivates the necessary further experimentation required for broader comparisons and conclusions.

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Section: Sf1 Family Helicasesmentioning

confidence: 99%

Section: Sf1 Family Helicasesmentioning

confidence: 99%

Section: Sf1 Family Helicasesmentioning

confidence: 99%

Section: Experimental Approaches and Mechanistic Modelsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

All motors have to decide is what to do with the DNA that is given them

Briggs

Fischer

2014

Biomolecular Concepts

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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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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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Escherichia coli RecBC helicase has two translocase activities controlled by a single ATPase motor

Cited by 49 publications

References 36 publications

All motors have to decide is what to do with the DNA that is given them

All motors have to decide is what to do with the DNA that is given them

How Does a Helicase Unwind DNA? Insights from RecBCD Helicase

How Chi Sequence Modifies RecBCD Single‐Stranded DNA Translocase Activity

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