Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes

Taylor, Andrew F.; Amundsen, Susan K.; Guttman, Miklós; Lee, Kelly K.; Luo, Jie; Ranish, Jeffrey A.; Smith, Gerald R.

doi:10.1016/j.jmb.2014.07.017

Cited by 41 publications

(61 citation statements)

References 51 publications

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“…Hence, both of these situations may mimic aspects of RecBCD after chi recognition. There have been suggestions that the RecB nuclease domain becomes repositioned after RecBCD recognizes a chi site[7, 58, 59]. In particular, the nuclease domain is involved in loading of RecA onto unwound ssDNA by directly binding to RecA[58].…”

Section: Discussionmentioning

confidence: 99%

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

Simon

Sokoloski

Hao

et al. 2016

Journal of Molecular Biology

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Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3′ to 5′ translocase, and RecD, a 5′ to 3′ translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair (bp) melting. However, some biochemical evidence suggests that DNA melting of multiple bp may occur separately from single stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity (RP) linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80 bp beyond the RP linkages. This ability requires an ATPase active RecB motor, the RecB “arm” domain and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD.

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Section: Discussionmentioning

confidence: 99%

“…Hence, the nuclease domain would need to move from its position in the crystal structure in order to interact with RecA[58]. Recently, Taylor et al[59] also presented evidence for a conformational change upon chi recognition that involves movement of the nuclease domain.…”

Section: Discussionmentioning

confidence: 99%

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

Simon

Sokoloski

Hao

et al. 2016

Journal of Molecular Biology

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“…The molecular basis for this intriguing result remains obscure. However, since the RecB nuclease domain is attached to the RecB motor by a long (≈70 amino acid) tether, and there is good evidence that the nuclease domain must detach from the position that it occupies in a crystal structure, there are many interesting possibilities that need to be tested.…”

Section: The Recb Arm May Function As a Double Stranded Dna Translocasementioning

confidence: 99%

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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“…We also showed that the secondary translocase of RecBC is not altered by chi recognition. Structural studies suggest that chi recognition induced nuclease domain movement, which consequently changes the degradation polarity and the ssDNA translocase activity of RecD …”

Section: Resultsmentioning

confidence: 99%

“…[4,5] Chi sequence thus allows RecBCD to discriminate the genome DNA of E. coli from foreign DNAs, as E. coli encoded thousands of copies of chi in its genome. [6,7] Upon chi recognition,R ecBCDs tarts to generate al ong3 ' ssDNA overhang terminiatedb yc hi and loads RecA to form an ucleoprotein filament in order to promote the homologousr ecombination. [8,9] The degradation preference of nucleased omain also shifts from 3'-strand to 5'-strand upon chi recognition.D egraded DNA fragments before chi recogni-tion is proposed as the substrates for Cas1-Cas2 complex, and is recently suggestedt ob ep reserved for future recognition of foreign DNA.…”

Section: Introductionmentioning

confidence: 99%

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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Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes

Cited by 41 publications

References 51 publications

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

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

How Does a Helicase Unwind DNA? Insights from RecBCD Helicase

How Chi Sequence Modifies RecBCD Single‐Stranded DNA Translocase Activity

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