Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

Amundsen, Susan K.; Taylor, Andrew F.; Smith, Gerald R.

doi:10.1038/s41598-020-73078-0

Cited by 11 publications

(4 citation statements)

References 47 publications

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“…Hence, when the two DNA binding sites in RecB and RecD are bound to ssDNA, the ADP nucleotide cofactor imposes a different degree of cooperativity, suggesting a different conformation and degree of interaction between RecB and RecD upon the different nucleotide cofactors. This may result from disrupting long-range intramolecular signaling between the three subunits as a response to different nucleotides ( 43 ).…”

Section: Resultsmentioning

confidence: 99%

Communication between DNA and nucleotide binding sites facilitates stepping by the RecBCD helicase

Gaydar,

Zananiri,

Saied

et al. 2024

Nucleic Acids Research

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Double-strand DNA breaks are the severest type of genomic damage, requiring rapid response to ensure survival. RecBCD helicase in prokaryotes initiates processive and rapid DNA unzipping, essential for break repair. The energetics of RecBCD during translocation along the DNA track are quantitatively not defined. Specifically, it's essential to understand the mechanism by which RecBCD switches between its binding states to enable its translocation. Here, we determine, by systematic affinity measurements, the degree of coupling between DNA and nucleotide binding to RecBCD. In the presence of ADP, RecBCD binds weakly to DNA that harbors a double overhang mimicking an unwinding intermediate. Consistently, RecBCD binds weakly to ADP in the presence of the same DNA. We did not observe coupling between DNA and nucleotide binding for DNA molecules having only a single overhang, suggesting that RecBCD subunits must both bind DNA to ‘sense’ the nucleotide state. On the contrary, AMPpNp shows weak coupling as RecBCD remains strongly bound to DNA in its presence. Detailed thermodynamic analysis of the RecBCD reaction mechanism suggests an ‘energetic compensation’ between RecB and RecD, which may be essential for rapid unwinding. Our findings provide the basis for a plausible stepping mechanism’ during the processive translocation of RecBCD.

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Section: Resultsmentioning

confidence: 99%

Communication between DNA and nucleotide binding sites facilitates stepping by the RecBCD helicase

Gaydar,

Zananiri,

Saied

et al. 2024

Nucleic Acids Research

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“…Whilst the biochemical and in vivo activities of RecBCD have been extensively studied ([11, 16, 17, 18, 19], and [7, 20] for review), less is known about the regulation of its expression. RecBCD has been reported to be expressed at very low levels [21, 22] and is present in less than ten molecules per cell [23].…”

Section: Introductionmentioning

confidence: 99%

An Hfq-dependent post-transcriptional mechanism fine tunes RecB expression inEscherichia coli

Kalita

Iosub

Granneman

et al. 2021

Preprint

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To preserve genome integrity, all living organisms have developed strategies to respond to chromosomal damage. One such response is the repair of DNA double-strand breaks (DSBs), one of the most toxic forms of DNA lesions. In E. coli, DSBs are repaired via the homologous recombination pathway, initiated by the RecBCD enzyme. RecBCD is essential for accurate chromosome maintenance but its over-expression can lead to reduced DNA repair ability. This apparent paradox suggests that RecBCD copy number may need to be tightly controlled within an optimal range. Using single-molecule fluorescence microscopy, we have established that RecB is present in very low abundance at mRNA and protein levels. RecB transcription shows high levels of fluctuations yet cell-to-cell protein variability remains remarkably low. We show that the post-transcriptional regulator Hfq binds to recBCD mRNAs and down-regulates RecB protein expression in vivo. Furthermore, when Hfq-mediated regulation is perturbed, we observe less effective noise reduction and reduced DNA repair capacity. Taken together, our results suggest a post-transcriptional regulatory mechanism where Hfq fine-tunes RecB expression by inhibiting RecB translation. This fine-tuning of RecB expression contributes to reducing noise in RecB protein expression and protects cells against the toxic consequences of too high RecBCD numbers.

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“…RecBCD then facilitates loading of RecA protein onto the resected 3' single strand resulting in a RecA filament that initiates homologous recombination. The RecB nuclease domain has been proposed to interact directly with RecA protein to facilitate its loading onto ssDNA [29].Since the proposed RecA binding site on the nuclease domain is occluded in the structures of RecBCD bound to DNA, it has been suggested that the nuclease domain is dynamic and may undock from the rest of RecBCD to facilitate RecA loading [29][30][31][32]. In fact, recent cryoEM structures of RecBCD bound to blunt-ended DNA show two classes of RecBCD-DNA structures [18].…”

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confidence: 99%

“…Since the proposed RecA binding site on the nuclease domain is occluded in the structures of RecBCD bound to DNA, it has been suggested that the nuclease domain is dynamic and may undock from the rest of RecBCD to facilitate RecA loading [29][30][31][32]. In fact, recent cryoEM structures of RecBCD bound to blunt-ended DNA show two classes of RecBCD-DNA structures [18].…”

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confidence: 99%

E. coliRecBCD Nuclease Domain Regulates Helicase Activity but not Single Stranded DNA Translocase Activity

Fazio,

Mersch,

Hao

et al. 2023

Preprint

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Much is still unknown about the mechanisms by which helicases unwind duplex DNA. Whereas structure-based models describe DNA unwinding as a consequence of mechanically pulling the DNA duplex across a wedge domain in the helicase by the single stranded (ss)DNA translocase activity of the ATPase motors, biochemical data indicate that processive DNA unwinding by the E. coli RecBCD helicase can occur in the absence of ssDNA translocation of the canonical RecB and RecD motors. Here, we present evidence that dsDNA unwinding is not a simple consequence of ssDNA translocation by the RecBCD motors. Using stopped-flow fluorescence approaches, we show that a RecB nuclease domain deletion variant (RecBΔNucCD) unwinds dsDNA at significantly slower rates than RecBCD, while the rate of ssDNA translocation is unaffected. This effect is primarily due to the absence of the nuclease domain and not the absence of the nuclease activity, since a nuclease-dead mutant, which retains the nuclease domain, showed no significant change in rates of ssDNA translocation or dsDNA unwinding relative to RecBCD on short DNA substrates (≤60 base pairs). This indicates that ssDNA translocation is not rate-limiting for DNA unwinding. RecBΔNucCD also initiates unwinding much slower than RecBCD from a blunt-ended DNA, although it binds with higher affinity than RecBCD. RecBΔNucCD also unwinds DNA ~two-fold slower than RecBCD on long DNA (~20 kilo base pair) in single molecule optical tweezer experiments, although the rates for RecBD1080ACD unwinding are intermediate between RecBCD and RecBΔNucCD. Surprisingly, significant pauses occur even in the absence of chi (crossover hotspot instigator) sites. We hypothesize that the nuclease domain influences the rate of DNA base pair melting, rather than DNA translocation, possibly allosterically. Since the rate of DNA unwinding by RecBCD also slows after it recognizes a chi sequence, RecBΔNucCD may mimic a post-chi state of RecBCD.

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Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

Cited by 11 publications

References 47 publications

Communication between DNA and nucleotide binding sites facilitates stepping by the RecBCD helicase

Communication between DNA and nucleotide binding sites facilitates stepping by the RecBCD helicase

An Hfq-dependent post-transcriptional mechanism fine tunes RecB expression inEscherichia coli

E. coliRecBCD Nuclease Domain Regulates Helicase Activity but not Single Stranded DNA Translocase Activity

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