scholarly journals Molecular characteristics of reiterative DNA unwinding by the Caenorhabditis elegans RecQ helicase

2019 ◽  
Vol 47 (18) ◽  
pp. 9708-9720 ◽  
Author(s):  
Seoyun Choi ◽  
Seung-Won Lee ◽  
Hajin Kim ◽  
Byungchan Ahn
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Protein A ◽  
Direct Interaction ◽  
Premature Aging ◽  
Molecular Characteristics ◽  
Dna Unwinding ◽  
Recq Helicase ◽  
Recq Helicases

Abstract The RecQ family of helicases is highly conserved both structurally and functionally from bacteria to humans. Defects in human RecQ helicases are associated with genetic diseases that are characterized by cancer predisposition and/or premature aging. RecQ proteins exhibit 3′-5′ helicase activity and play critical roles in genome maintenance. Recent advances in single-molecule techniques have revealed the reiterative unwinding behavior of RecQ helicases. However, the molecular mechanisms involved in this process remain unclear, with contradicting reports. Here, we characterized the unwinding dynamics of the Caenorhabditis elegans RecQ helicase HIM-6 using single-molecule fluorescence resonance energy transfer measurements. We found that HIM-6 exhibits reiterative DNA unwinding and the length of DNA unwound by the helicase is sharply defined at 25–31 bp. Experiments using various DNA substrates revealed that HIM-6 utilizes the mode of ‘sliding back’ on the translocated strand, without strand-switching for rewinding. Furthermore, we found that Caenorhabditis elegans replication protein A, a single-stranded DNA binding protein, suppresses the reiterative behavior of HIM-6 and induces unidirectional, processive unwinding, possibly through a direct interaction between the proteins. Our findings shed new light on the mechanism of DNA unwinding by RecQ family helicases and their co-operation with RPA in processing DNA.

scholarly journals RecQ helicases: suppressors of tumorigenesis and premature aging

2003 ◽  
Vol 374 (3) ◽  
pp. 577-606 ◽  
Author(s):  
Csanád Z. BACHRATI ◽  
Ian D. HICKSON
Keyword(s):  
Replication Fork ◽  
Germ Line ◽  
Genetic Disorders ◽  
Premature Aging ◽  
Recq Helicase ◽  
Dna Helicases ◽  
Recq Helicases ◽  
Protein Partners ◽  
Line Defects ◽  
Rothmund Thomson Syndrome

The RecQ helicases represent a subfamily of DNA helicases that are highly conserved in evolution. Loss of RecQ helicase function leads to a breakdown in the maintenance of genome integrity, in particular hyper-recombination. Germ-line defects in three of the five known human RecQ helicases give rise to defined genetic disorders associated with cancer predisposition and/or premature aging. These are Bloom's syndrome, Werner's syndrome and Rothmund–Thomson syndrome, which are caused by defects in the genes BLM, WRN and RECQ4 respectively. Here we review the properties of RecQ helicases in organisms from bacteria to humans, with an emphasis on the biochemical functions of these enzymes and the range of protein partners that they operate with. We will discuss models in which RecQ helicases are required to protect against replication fork demise, either through prevention of fork breakdown or restoration of productive DNA synthesis.

scholarly journals Single-molecule visualization of human BLM helicase as it acts upon double- and single-stranded DNA substrates

2019 ◽  
Vol 47 (21) ◽  
pp. 11225-11237 ◽  
Author(s):  
Chaoyou Xue ◽  
James M Daley ◽  
Xiaoyu Xue ◽  
Justin Steinfeld ◽  
Youngho Kwon ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Genetic Disorder ◽  
Protein A ◽  
Base Pairs ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Blm Helicase ◽  
Dna Replication And Repair ◽  
Regulatory Effects

Abstract Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70–80 base pairs per second) unwinding extensive tracts (∼8–10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.

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

2015 ◽  
Vol 112 (50) ◽  
pp. E6852-E6861 ◽  
Author(s):  
Behzad Rad ◽  
Anthony L. Forget ◽  
Ronald J. Baskin ◽  
Stephen C. Kowalczykowski
Keyword(s):  
Single Molecule ◽  
Fluorescent Sensor ◽  
Holliday Junctions ◽  
Dna Unwinding ◽  
Recq Helicase ◽  
Dna Breaks ◽  
Dna Helicases ◽  
Double Stranded Dna ◽  
Functional Forms ◽  
And Migration

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.

scholarly journals Human RPA activates BLM’s bidirectional DNA unwinding from a nick

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Zhenheng Qin ◽  
Lulu Bi ◽  
Xi-Miao Hou ◽  
Siqi Zhang ◽  
Xia Zhang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Single Molecule ◽  
Genome Stability ◽  
Protein A ◽  
Replication Protein A ◽  
Replication Protein ◽  
High Abundance ◽  
Dna Unwinding ◽  
Dna Replication And Repair

BLM is a multifunctional helicase that plays critical roles in maintaining genome stability. It processes distinct DNA substrates, but not nicked DNA, during many steps in DNA replication and repair. However, how BLM prepares itself for diverse functions remains elusive. Here, using a combined single-molecule approach, we find that a high abundance of BLMs can indeed unidirectionally unwind dsDNA from a nick when an external destabilizing force is applied. Strikingly, human replication protein A (hRPA) not only ensures that limited quantities of BLMs processively unwind nicked dsDNA under a reduced force but also permits the translocation of BLMs on both intact and nicked ssDNAs, resulting in a bidirectional unwinding mode. This activation necessitates BLM targeting on the nick and the presence of free hRPAs in solution whereas direct interactions between them are dispensable. Our findings present novel DNA unwinding activities of BLM that potentially facilitate its function switching in DNA repair.

scholarly journals Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

2017 ◽  
Vol 114 (4) ◽  
pp. E466-E475 ◽  
Author(s):  
Gábor M. Harami ◽  
Yeonee Seol ◽  
Junghoon In ◽  
Veronika Ferencziová ◽  
Máté Martina ◽  
...  
Keyword(s):  
Single Molecule ◽  
Domain Architecture ◽  
Dna Recombination ◽  
Recq Helicase ◽  
Dna Structures ◽  
Recq Helicases ◽  
Differential Recognition ◽  
D Loop ◽  
Mechanistic Basis

Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show thatEscherichia coliRecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.

scholarly journals The HRDC domain oppositely modulates the unwinding activity of E. coli RecQ helicase on duplex DNA and G-quadruplex

2020 ◽  
Vol 295 (51) ◽  
pp. 17646-17658
Author(s):  
Fang-Yuan Teng ◽  
Ting-Ting Wang ◽  
Hai-Lei Guo ◽  
Ben-Ge Xin ◽  
Bo Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Stability ◽  
Premature Aging ◽  
Binding Modes ◽  
Ssdna Binding ◽  
Recq Helicases ◽  
E Coli ◽  
Single Molecule Fret ◽  
G4 Dna ◽  
Long Time

RecQ family helicases are highly conserved from bacteria to humans and have essential roles in maintaining genome stability. Mutations in three human RecQ helicases cause severe diseases with the main features of premature aging and cancer predisposition. Most RecQ helicases shared a conserved domain arrangement which comprises a helicase core, an RecQ C-terminal domain, and an auxiliary element helicase and RNaseD C-terminal (HRDC) domain, the functions of which are poorly understood. In this study, we systematically characterized the roles of the HRDC domain in E. coli RecQ in various DNA transactions by single-molecule FRET. We found that RecQ repetitively unwinds the 3′-partial duplex and fork DNA with a moderate processivity and periodically patrols on the ssDNA in the 5′-partial duplex by translocation. The HRDC domain significantly suppresses RecQ activities in the above transactions. In sharp contrast, the HRDC domain is essential for the deep and long-time unfolding of the G4 DNA structure by RecQ. Based on the observations that the HRDC domain dynamically switches between RecA core- and ssDNA-binding modes after RecQ association with DNA, we proposed a model to explain the modulation mechanism of the HRDC domain. Our findings not only provide new insights into the activities of RecQ on different substrates but also highlight the novel functions of the HRDC domain in DNA metabolisms.

scholarly journals Single-molecule level structural dynamics of DNA unwinding by human mitochondrial Twinkle helicase

2020 ◽  
Vol 295 (17) ◽  
pp. 5564-5576 ◽  
Author(s):  
Parminder Kaur ◽  
Matthew J. Longley ◽  
Hai Pan ◽  
Wendy Wang ◽  
Preston Countryman ◽  
...  
Keyword(s):  
Dna Binding ◽  
Mitochondrial Genome ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Dna Unwinding ◽  
Microscopy Imaging ◽  
Single Molecule Level ◽  
Closed Ring ◽  
Mtdna Replication

Knowledge of the molecular events in mitochondrial DNA (mtDNA) replication is crucial to understanding the origins of human disorders arising from mitochondrial dysfunction. Twinkle helicase is an essential component of mtDNA replication. Here, we employed atomic force microscopy imaging in air and liquids to visualize ring assembly, DNA binding, and unwinding activity of individual Twinkle hexamers at the single-molecule level. We observed that the Twinkle subunits self-assemble into hexamers and higher-order complexes that can switch between open and closed-ring configurations in the absence of DNA. Our analyses helped visualize Twinkle loading onto and unloading from DNA in an open-ringed configuration. They also revealed that closed-ring conformers bind and unwind several hundred base pairs of duplex DNA at an average rate of ∼240 bp/min. We found that the addition of mitochondrial single-stranded (ss) DNA–binding protein both influences the ways Twinkle loads onto defined DNA substrates and stabilizes the unwound ssDNA product, resulting in a ∼5-fold stimulation of the apparent DNA-unwinding rate. Mitochondrial ssDNA-binding protein also increased the estimated translocation processivity from 1750 to >9000 bp before helicase disassociation, suggesting that more than half of the mitochondrial genome could be unwound by Twinkle during a single DNA-binding event. The strategies used in this work provide a new platform to examine Twinkle disease variants and the core mtDNA replication machinery. They also offer an enhanced framework to investigate molecular mechanisms underlying deletion and depletion of the mitochondrial genome as observed in mitochondrial diseases.

scholarly journals A role for the fusogen eff-1 in epidermal stem cell number robustness in Caenorhabditis elegans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sneha L. Koneru ◽  
Fu Xiang Quah ◽  
Ritobrata Ghose ◽  
Mark Hintze ◽  
Nicola Gritti ◽  
...  
Keyword(s):  
Stem Cell ◽  
Caenorhabditis Elegans ◽  
Cell Fate ◽  
Single Molecule ◽  
Phenotypic Variability ◽  
Low Frequency ◽  
Time Lapse ◽  
Cell Number ◽  
Seam Cell ◽  
Neuronal Tissues

AbstractDevelopmental patterning in Caenorhabditis elegans is known to proceed in a highly stereotypical manner, which raises the question of how developmental robustness is achieved despite the inevitable stochastic noise. We focus here on a population of epidermal cells, the seam cells, which show stem cell-like behaviour and divide symmetrically and asymmetrically over post-embryonic development to generate epidermal and neuronal tissues. We have conducted a mutagenesis screen to identify mutants that introduce phenotypic variability in the normally invariant seam cell population. We report here that a null mutation in the fusogen eff-1 increases seam cell number variability. Using time-lapse microscopy and single molecule fluorescence hybridisation, we find that seam cell division and differentiation patterns are mostly unperturbed in eff-1 mutants, indicating that cell fusion is uncoupled from the cell differentiation programme. Nevertheless, seam cell losses due to the inappropriate differentiation of both daughter cells following division, as well as seam cell gains through symmetric divisions towards the seam cell fate were observed at low frequency. We show that these stochastic errors likely arise through accumulation of defects interrupting the continuity of the seam and changing seam cell shape, highlighting the role of tissue homeostasis in suppressing phenotypic variability during development.

scholarly journals Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

2021 ◽  
Vol 22 (5) ◽  
pp. 2398
Author(s):  
Wooyoung Kang ◽  
Seungha Hwang ◽  
Jin Young Kang ◽  
Changwon Kang ◽  
Sungchul Hohng
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Facilitated Diffusion ◽  
One Dimensional ◽  
Bacterial Transcription ◽  
Fluorescence Measurements ◽  
Dimensional Diffusion ◽  
Single Molecule Studies ◽  
Hopping Diffusion

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

scholarly journals Infrared nanospectroscopy reveals the molecular interaction fingerprint of an aggregation inhibitor with single Aβ42 oligomers

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Francesco Simone Ruggeri ◽  
Johnny Habchi ◽  
Sean Chia ◽  
Robert I. Horne ◽  
Michele Vendruscolo ◽  
...  
Keyword(s):  
Small Molecules ◽  
Single Molecule ◽  
Protein Misfolding ◽  
Molecular Mechanisms ◽  
Chemical Sensitivity ◽  
Incomplete Knowledge ◽  
Parkinson’S Diseases ◽  
Aggregation Inhibitor ◽  
Misfolding Diseases

AbstractSignificant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer’s and Parkinson’s diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the structure and interaction between single Aβ42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, which is able to prevent Aβ42 aggregation in vitro and reverses its neurotoxicity in cell and animal models of Alzheimer’s disease. Our results demonstrate that the carboxyl group of this compound interacts with Aβ42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as a powerful tool in structure-based drug discovery for protein misfolding diseases.

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