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Journal ArticleDOI

Fork sensing and strand switching control antagonistic activities of RecQ helicases

TL;DR: This work investigates the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECZ3 at the single-molecule level using magnetic tweezers and provides a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family.
Abstract: RecQ helicases have essential roles in maintaining genome stability during replication and in controlling double-strand break repair by homologous recombination. Little is known about how the different RecQ helicases found in higher eukaryotes achieve their specialized and partially opposing functions. Here, we investigate the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECQ3 at the single-molecule level using magnetic tweezers. Although AtRECQ2 predominantly unwinds forked DNA substrates in a highly repetitive fashion, AtRECQ3 prefers to rewind, that is, to close preopened DNA forks. For both enzymes, this process is controlled by frequent strand switches and active sensing of the unwinding fork. The relative extent of the strand switches towards unwinding or towards rewinding determines the predominant direction of the enzyme. Our results provide a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family.

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Citations
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Journal ArticleDOI
TL;DR: It is proposed that, in addition to the ssDNA site on the protein that interacts with the translocating strand, Pif1 has a second site that binds the 3'-ssDNA of the substrate, which modulates the degree to which re-winding counteracts unwinding.

19 citations

Journal ArticleDOI
TL;DR: The data suggest that the dominant and repetitive mode of DNA opening of the helicase can be used to allow efficient DNA replication, with DNA synthesis on the nontranslocating strand rectifying the DNA unwinding activity.
Abstract: Members of the Pif1 family of helicases function in multiple pathways that involve DNA synthesis: DNA replication across G-quadruplexes; break-induced replication; and processing of long flaps during Okazaki fragment maturation. Furthermore, Pif1 increases strand-displacement DNA synthesis by DNA polymerase δ and allows DNA replication across arrays of proteins tightly bound to DNA. This is a surprising feat since DNA rewinding or annealing activities limit the amount of single-stranded DNA product that Pif1 can generate, leading to an apparently poorly processive helicase. In this work, using single-molecule Forster resonance energy transfer approaches, we show that 2 members of the Pif1 family of helicases, Pif1 from Saccharomyces cerevisiae and Pfh1 from Schizosaccharomyces pombe, unwind double-stranded DNA by a branched mechanism with 2 modes of activity. In the dominant mode, only short stretches of DNA can be processively and repetitively opened, with reclosure of the DNA occurring by mechanisms other than strand-switching. In the other less frequent mode, longer stretches of DNA are unwound via a path that is separate from the one leading to repetitive unwinding. Analysis of the kinetic partitioning between the 2 different modes suggests that the branching point in the mechanism is established by conformational selection, controlled by the interaction of the helicase with the 3' nontranslocating strand. The data suggest that the dominant and repetitive mode of DNA opening of the helicase can be used to allow efficient DNA replication, with DNA synthesis on the nontranslocating strand rectifying the DNA unwinding activity.

18 citations


Cites background from "Fork sensing and strand switching c..."

  • ...Strandswitching during unwinding has been proposed for different DNA helicases as a mechanism that leads to rezipping of the opened dsDNA (7, 11, 50)....

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  • ...For example, strand-switching during unwinding, with the helicase being able to jump to the opposite ssDNA strand and translocate back, has been proposed for multiple helicases (7, 11, 50, 55), including Pif1 (47)....

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Journal ArticleDOI
TL;DR: This work reviews the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins and uncovered features that were obscured by bulk studies.
Abstract: Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase-induced re-hybridization and stalled fork rescue. This article is protected by copyright. All rights reserved.

17 citations


Cites background from "Fork sensing and strand switching c..."

  • ...As the enzyme passes the apex, the force applied by the closing fork induces the enzyme to switch strands reverting into an unwinding mode.(58) The rate of DNA unwinding by helicases has also been studied as a function of the local AT (or GC) content of the hairpin....

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Journal ArticleDOI
10 Dec 2019-Genes
TL;DR: Gaining knowledge about plant helicases, their interplay, as well as the manipulation of their pathways, possesses the potential for improving agriculture and might even help to cope with the increasing obstacles of climate change threatening food security in completely new ways.
Abstract: Genetic information of all organisms is coded in double-stranded DNA. DNA helicases are essential for unwinding this double strand when it comes to replication, repair or transcription of genetic information. In this review, we will focus on what is known about a variety of DNA helicases that are required to ensure genome stability in plants. Due to their sessile lifestyle, plants are especially exposed to harmful environmental factors. Moreover, many crop plants have large and highly repetitive genomes, making them absolutely dependent on the correct interplay of DNA helicases for safeguarding their stability. Although basic features of a number of these enzymes are conserved between plants and other eukaryotes, a more detailed analysis shows surprising peculiarities, partly also between different plant species. This is additionally of high relevance for plant breeding as a number of these helicases are also involved in crossover control during meiosis and influence the outcome of different approaches of CRISPR/Cas based plant genome engineering. Thus, gaining knowledge about plant helicases, their interplay, as well as the manipulation of their pathways, possesses the potential for improving agriculture. In the long run, this might even help us cope with the increasing obstacles of climate change threatening food security in completely new ways.

14 citations


Cites background from "Fork sensing and strand switching c..."

  • ...While both show a highly repetitive DNA helicase function, AtRECQ2 preferentially unwinds DNA but AtRECQ3 predominantly rewinds the DNA after short unwinding activity [34]....

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Book ChapterDOI
TL;DR: A practical guide to applying single-molecule optical-trapping techniques to study the dynamics of individual proteins in the bacteriophage T7 replisome, as well as the coordination among them.
Abstract: The replisome is a multiprotein molecular machinery responsible for the replication of DNA It is composed of several specialized proteins each with dedicated enzymatic activities, and in particular, helicase unwinds double-stranded DNA and DNA polymerase catalyzes the synthesis of DNA Understanding how a replisome functions in the process of DNA replication requires methods to dissect the mechanisms of individual proteins and of multiproteins acting in concert Single-molecule optical-trapping techniques have proved to be a powerful approach, offering the unique ability to observe and manipulate biomolecules at the single-molecule level and providing insights into the mechanisms of molecular motors and their interactions and coordination in a complex Here, we describe a practical guide to applying these techniques to study the dynamics of individual proteins in the bacteriophage T7 replisome, as well as the coordination among them We also summarize major findings from these studies, including nucleotide-specific helicase slippage and new lesion bypass pathway in T7 replication

14 citations


Additional excerpts

  • ...Slippage has been observed with other helicases, however, it appears to result from different causes (Klaue et al., 2013; Lee et al., 2014; Manosas, Spiering, Ding, Croquette, & Benkovic, 2012; Myong, Bruno, Pyle, & Ha, 2007; Myong, Rasnik, Joo, Lohman, & Ha, 2005; Qi, Pugh, Spies, & Chemla, 2013;…...

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References
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Journal ArticleDOI
TL;DR: This review sets out to define a nomenclature for helicase and translocase enzymes based on current knowledge of sequence, structure, and mechanism, and delineate six superfamilies of enzymes, with examples of crystal structures where available.
Abstract: Helicases and translocases are a ubiquitous, highly diverse group of proteins that perform an extraordinary variety of functions in cells. Consequently, this review sets out to define a nomenclature for these enzymes based on current knowledge of sequence, structure, and mechanism. Using previous definitions of helicase families as a basis, we delineate six superfamilies of enzymes, with examples of crystal structures where available, and discuss these structures in the context of biochemical data to outline our present understanding of helicase and translocase activity. As a result, each superfamily is subdivided, where appropriate, on the basis of mechanistic understanding, which we hope will provide a framework for classification of new superfamily members as they are discovered and characterized.

1,145 citations

Journal ArticleDOI
02 Apr 1999-Cell
TL;DR: Two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex are determined, providing snapshots of different steps on the catalytic pathway, providing evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism.

759 citations

Journal ArticleDOI
TL;DR: The structure of the HCV NS3 RNA helicase domain complexed with a single-stranded DNA oligonucleotide has been solved to 2.2 A resolution and is a member of a superfamily of helicases, termed superfamily II.

630 citations

Journal ArticleDOI
TL;DR: This Review discusses how these proteins might suppress genomic rearrangements, and therefore function as 'caretaker' tumour suppressors.
Abstract: Around 1% of the open reading frames in the human genome encode predicted DNA and RNA helicases. One highly conserved group of DNA helicases is the RecQ family. Genetic defects in three of the five human RecQ helicases, BLM, WRN and RECQ4, give rise to defined syndromes associated with cancer predisposition, some features of premature ageing and chromosomal instability. In recent years, there has been a tremendous advance in our understanding of the cellular functions of individual RecQ helicases. In this Review, we discuss how these proteins might suppress genomic rearrangements, and therefore function as 'caretaker' tumour suppressors.

432 citations

Journal ArticleDOI
29 Dec 2006-Cell
TL;DR: A series of crystal structures of the UvrD helicase complexed with DNA and ATP hydrolysis intermediates reveal that ATP binding alone leads to unwinding of 1 base pair by directional rotation and translation of the DNA duplex, and ADP and Pi release leads to translocation of the developing single strand.

338 citations