Visualizing helicases unwinding DNA at the single molecule level

Fili, Natalia; Mashanov, Gregory I.; Toseland, Christopher P.; Batters, Christopher; Wallace, Mark I.; Yeeles, Joseph T.P.; Dillingham, Mark S.; Webb, Martin R.; Molloy, Justin E.

doi:10.1093/nar/gkq173

Cited by 52 publications

(54 citation statements)

References 45 publications

(78 reference statements)

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“…Previous AddAB DNA unwinding measurements have uncovered static disorder and pausing during translocation, but the effect of Chi sequences has never been directly investigated (32,33). In this work, we explored the effect of Chi recognition on AddAB translocation at the single-molecule level, using magnetic tweezers.…”

Section: Discussionmentioning

confidence: 99%

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On the mechanism of recombination hotspot scanning during double-stranded DNA break resection

Carrasco

Gilhooly

Dillingham

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Double-stranded DNA break repair by homologous recombination is initiated by resection of free DNA ends to produce a 3′-ssDNA overhang. In bacteria, this reaction is catalyzed by helicase-nuclease complexes such as AddAB in a manner regulated by specific recombination hotspot sequences called Crossover hotspot instigator (Chi). We have used magnetic tweezers to investigate the dynamics of AddAB translocation and hotspot scanning during double-stranded DNA break resection. AddAB was prone to stochastic pausing due to transient recognition of Chi-like sequences, unveiling an antagonistic relationship between DNA translocation and sequence-specific DNA recognition. Pauses at bona fide Chi sequences were longer, were nonexponentially distributed, and resulted in an altered velocity upon restart of translocation downstream of Chi. We propose a model for the recognition of Chi sequences to explain the origin of pausing during failed and successful hotspot recognition.protein motor | single molecule biophysics | DNA-end processing | real-time measurements | protein-DNA interactions D ouble-stranded DNA breaks (DSBs) are formed frequently, both as a result of exogenous and endogenous DNA damaging agents and as intermediates in programmed DNA rearrangements. Failure to properly repair DSBs results in loss of chromosome structural integrity and genomic instability and is associated with developmental defects, deficiencies of the immune system, and cancer predisposition. There are multiple mechanisms for DSB repair, but faithful repair generally requires the homologous recombination pathway, which can occur only if a suitable donor molecule such as the sister chromatid is available (1). Recombinational repair of DSBs is initiated by the longrange resection of the DNA end to form a 3′-terminated ssDNA overhang that acts as a substrate for the RecA/Rad51 recombinase. In all domains of life, this reaction is catalyzed by an array of helicases and nucleases but is best characterized in bacteria where either an AddAB-or a RecBCD-type helicase-nuclease complex recognizes the DNA end structure and then promotes its processive unwinding and concomitant resection (2, 3). A third class of helicase-nuclease named AdnAB is found in the mycobacterial niche and it shares limited structural similarity with AddAB enzymes (4). An apparently unique feature of the DNA break processing in bacteria is its control by cisacting DNA sequences called Crossover hotspot instigator (Chi) sequences. In the absence of hotspot sequences, AddAB or RecBCD complexes processively and rapidly degrade DNA in an ATP-dependent fashion, a mode of action that probably acts to degrade foreign DNA or in the restart of regressed replication forks (5). Recognition of Chi sequences by the translocating enzymes has many different effects on AddAB and/or RecBCD complexes, all of which serve to promote downstream recombination (6). These include the attenuation of the nuclease activity downstream of Chi on the 3′ strand (7), the promotion of DNA unwinding (8), and the lo...

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Section: Discussionmentioning

confidence: 99%

“…Biotinylated AddAB and AddAB F210A were purified as described previously (32). DNA molecules for single-molecule experiments were produced by PCR, using one digoxigenin (DIG)-labeled primer (distal end), one unlabeled primer (proximal end), and a high-fidelity DNA polymerase.…”

Section: Methodsmentioning

confidence: 99%

On the mechanism of recombination hotspot scanning during double-stranded DNA break resection

Carrasco

Gilhooly

Dillingham

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Fluorescence techniques coupled with single-molecule microscopy have emerged as a powerful method for studying the unwinding mechanism of helicases (30)(31)(32)(33)(34)(35). These assays measure individual enzymes directly or indirectly through their actions on individual DNA molecules, thus alleviating many of the drawbacks of ensemble experiments.…”

Section: Significancementioning

confidence: 99%

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Rad

Forget

Baskin

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40-60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.recombination | fluorescence | TIRF microscopy | DNA repair | BLM

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“…The assays can be performed in real-time, in solution, with high-time resolution and sensitivity down to single molecules. This has allowed the measurement of protein interactions, enzymatic activity, conformation changes, localization of proteins, and the ability to see individual proteins moving in real time [2,8,20,42,51,59].…”

Section: Introductionmentioning

confidence: 99%

Fluorescent labeling and modification of proteins

2013

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This review provides an outline for fluorescent labeling of proteins. Fluorescent assays are very diverse providing the most sensitive and robust methods for observing biological processes. Here, different types of labels and methods of attachment are discussed in combination with their fluorescent properties. The advantages and disadvantages of these different methods are highlighted, allowing the careful selection for different applications, ranging from ensemble spectroscopy assays through to single-molecule measurements.

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Visualizing helicases unwinding DNA at the single molecule level

Cited by 52 publications

References 45 publications

On the mechanism of recombination hotspot scanning during double-stranded DNA break resection

On the mechanism of recombination hotspot scanning during double-stranded DNA break resection

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Fluorescent labeling and modification of proteins

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