122 results found
Anand R, Buechelmaier E, Belan O, et al., 2021, HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51, NATURE, Vol: 601, Pages: 268-+, ISSN: 0028-0836
In recent years, fluorogenic RNA aptamers, such as Spinach, Broccoli, Corn, Mango, Coral, and Pepper have gathered traction as an efficient alternative labeling strategy for background-free imaging of cellular RNAs. However, their application has been somewhat limited by relatively inefficient folding and fluorescent stability. With the recent advent of novel RNA-Mango variants which are improved in both fluorescence intensity and folding stability in tandem arrays, it is now possible to image RNAs with single-molecule sensitivity. Here we discuss the protocol for imaging Mango II tagged RNAs in both fixed and live cells.
Newton MD, Fairbanks SD, Thomas JA, et al., 2021, A Minimal Load‐and‐Lock Ru II Luminescent DNA Probe, Angewandte Chemie, Vol: 133, Pages: 21120-21127, ISSN: 0044-8249
Losito M, Smith QM, Newton MD, et al., 2021, Cas12a target search and cleavage on force-stretched DNA, PHYSICAL CHEMISTRY CHEMICAL PHYSICS, Vol: 23, ISSN: 1463-9076
Newton MD, Fairbanks SD, Thomas JA, et al., 2021, A minimal load-and-lock Ru-II luminescent DNA probe, Angewandte Chemie International Edition, Vol: 60, Pages: 20952-20959, ISSN: 1433-7851
Threading intercalators bind DNA with high affinities. Here, we describe single-molecule studies on a cell-permeant luminescent dinuclear ruthenium(II) complex that has been previously shown to thread only into short, unstable duplex structures. Using optical tweezers and confocal microscopy, we show that this complex threads and locks into force-extended duplex DNA in a two-step mechanism. Detailed kinetic studies reveal that an individual stereoisomer of the complex exhibits the highest binding affinity reported for such a mono-intercalator. This stereoisomer better preserves the biophysical properties of DNA than the widely used SYTOX Orange. Interestingly, threading into torsionally constrained DNA decreases dramatically, but is rescued on negatively supercoiled DNA. Given the “light-switch” properties of this complex on binding DNA, it can be readily used as a long-lived luminescent label for duplex or negatively supercoiled DNA through a unique “load-and-lock” protocol.
Boulton S, Anand R, Buechelmaier E, et al., 2021, HELQ is a dual function DSB repair enzyme modulated by RPA and RAD51
<jats:title>Abstract</jats:title> <jats:p>DNA double strand breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3’ to 5’ polarity, whose disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2-4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C and, persistence of RAD51 foci upon DNA damage3,5. Notably, HELQ binds to RPA and the RAD51 paralog BCDX2 complex but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here, we report that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry and single-molecule imaging (SMI), we establish that RAD51 forms a co-complex with and strongly stimulates HELQ as it translocates during DNA unwinding. Conversely, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary strands. Finally, we show that HELQ deficiency in cells compromises single-strand annealing (SSA) and microhomology-mediated end joining (MMEJ) pathways and increases long-tract gene conversion tracts (LTGC) during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair by virtue of co-factor dependent modulation of intrinsic translocase and DNA strand annealing activities.</jats:p>
Belan O, Moore G, Kaczmarczyk A, et al., 2021, Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis., STAR Protocols, Vol: 2, Pages: 1-18, ISSN: 2666-1667
Here, we describe a rapid and versatile protocol to generate gapped DNA substrates for single-molecule (SM) analysis using optical tweezers via site-specific Cas9 nicking and force-induced melting. We provide examples of single-stranded (ss) DNA gaps of different length and position. We outline protocols to visualize these substrates by replication protein A-enhanced Green Fluorescent Protein (RPA-eGFP) and SYTOX Orange staining using commercially available optical tweezers (C-TRAP). Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021).
Belan O, Barroso C, Kaczmarczyk A, et al., 2021, Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins, Molecular Cell, Vol: 81, Pages: 1058-1073.e7, ISSN: 1097-2765
Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a “chaperone” to promote 3′ to 5′ filament growth via highly dynamic engagement with 5′ filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.
Iino H, Abdolahzadeh A, Dolgosheina E, et al., 2021, Rapid Clinical Diagnostic Viral Detection with Saliva by a Novel Single Step Nested Mango-NASBA Assay, 65th Annual Meeting of the Biophysical-Society (BPS), Publisher: CELL PRESS, Pages: 196A-196A, ISSN: 0006-3495
Belan O, Barroso C, Kaczmarczyk A, et al., 2021, Single-Molecule Investigation of Rad-51 Presynaptic Filament Assembly and the Role of Mediator Proteins, 65th Annual Meeting of the Biophysical-Society (BPS), Publisher: CELL PRESS, Pages: 33A-33A, ISSN: 0006-3495
Cawte AD, Unrau PJ, Rueda DS, 2020, Live cell imaging of single RNA molecules with fluorogenic Mango II arrays, Nature Communications, Vol: 11, ISSN: 2041-1723
RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to bleached fluorophore replacement. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.
Gutierrez-Escribano P, Newton MD, Llauro A, et al., 2019, A conserved ATP- and Scc2/4-dependent activity for cohesin in tethering DNA molecules, Science Advances, Vol: 5, Pages: 1-15, ISSN: 2375-2548
Sister chromatid cohesion requires cohesin to act as a protein linker to hold chromatids together. How cohesin tethers chromatids remains poorly understood. We have used optical tweezers to visualize cohesin as it holds DNA molecules. We show that cohesin complexes tether DNAs in the presence of Scc2/Scc4 and ATP demonstrating a conserved activity from yeast to humans. Cohesin forms two classes of tethers: a “permanent bridge” resisting forces over 80 pN and a force-sensitive “reversible bridge.” The establishment of bridges requires physical proximity of dsDNA segments and occurs in a single step. “Permanent” cohesin bridges slide when they occur in trans, but cannot be removed when in cis. Therefore, DNAs occupy separate physical compartments in cohesin molecules. We finally demonstrate that cohesin tetramers can compact linear DNA molecules stretched by very low force (below 1 pN), consistent with the possibility that, like condensin, cohesin is also capable of loop extrusion.
Miura M, Dey S, Ramanayake S, et al., 2019, Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modelling, PLoS Pathogens, Vol: 15, ISSN: 1553-7366
The human T cell leukemia virus HTLV-1 establishes a persistent infection in vivo in which the viral sense-strand transcription is usually silent at a given time in each cell. However, cellular stress responses trigger the reactivation of HTLV-1, enabling the virus to transmit to a new host cell. Using single-molecule RNA FISH, we measured the kinetics of the HTLV-1 transcriptional reactivation in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1+ individuals. The abundance of the HTLV-1 sense and antisense transcripts was quantified hourly during incubation of the HTLV-1-infected PBMCs ex vivo. We found that, in each cell, the sense-strand transcription occurs in two distinct phases: the initial low-rate transcription is followed by a phase of rapid transcription. The onset of transcription peaked between 1 and 3 hours after the start of in vitro incubation. The variance in the transcription intensity was similar in polyclonal HTLV-1+ PBMCs (with tens of thousands of distinct provirus insertion sites), and in samples with a single dominant HTLV-1+ clone. A stochastic simulation model was developed to estimate the parameters of HTLV-1 proviral transcription kinetics. In PBMCs from a leukemic subject with one dominant T-cell clone, the model indicated that the average duration of HTLV-1 sense-strand activation by Tax (i.e. the rapid transcription) was less than one hour. HTLV-1 antisense transcription was stable during reactivation of the sense-strand. The antisense transcript HBZ was produced at an average rate of ~0.1 molecules per hour per HTLV-1+ cell; however, between 20% and 70% of HTLV-1-infected cells were HBZ-negative at a given time, the percentage depending on the individual subject. HTLV-1-infected cells are exposed to a range of stresses when they are drawn from the host, which initiate the viral reactivation. We conclude that whereas antisense-strand transcription is stable throughout the stress response, the HTLV-1 sense-strand reactivati
Cawte AD, Unrau PJ, Rueda DS, 2019, Live Cell Imaging of Single RNA Molecules with Fluorogenic Mango II Arrays, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p>RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (β-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to fluorophore exchange. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.</jats:p>
Wilson MD, Renault L, Maskell DP, et al., 2019, Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer, Nature Communications, Vol: 10, ISSN: 2041-1723
Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the histone octamer at the site of integration. To clarify the mechanism of DNA looping by integrase, we determined a 3.9 Å resolution structure of the prototype foamy virus intasome engaged with a nucleosome core particle. The structural data along with complementary single-molecule Förster resonance energy transfer measurements reveal twisting and sliding of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA by approximately two base pairs along the histone octamer accommodates the necessary DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for nucleosomal DNA repositioning, bearing unexpected similarities to chromatin remodelers.
Bruno L, Ramlall V, Studer RA, et al., 2019, Selective deployment of transcription factor paralogs with submaximal strength facilitates gene regulation in the immune system, Nature Immunology, Vol: 20, Pages: 1372-1380, ISSN: 1529-2908
In multicellular organisms, duplicated genes can diverge through tissue-specific gene expression patterns, as exemplified by highly regulated expression of Runx transcription factor paralogs with apparent functional redundancy. Here we asked what cell type-specific biologies might be supported by the selective expression of Runx paralogs during Langerhans cell and inducible regulatory T cell differentiation. We uncovered functional non-equivalence between Runx paralogs. Selective expression of native paralogs allowed integration of transcription factor activity with extrinsic signals, while non-native paralogs enforced differentiation even in the absence of exogenous inducers. DNA-binding affinity was controlled by divergent amino acids within the otherwise highly conserved RUNT domain, and evolutionary reconstruction suggested convergence of RUNT domain residues towards sub-maximal strength. Hence, the selective expression of gene duplicates in specialized cell types can synergize with the acquisition of functional differences to enable appropriate gene expression, lineage choice and differentiation in the mammalian immune system.
CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.
Newton M, Taylor B, Driessen R, et al., 2018, DNA stretching induces Cas9 off-target binding and cleavage, Nature Structural and Molecular Biology, ISSN: 1545-9985
CRISPR/Cas9 is a powerful genome editing tool, but spurious off-target edits present a barrier towards therapeutic applications. To understand how CRISPR/Cas9 discrimi-nates between on- and off-targets, we have developed a single-molecule assay com-bining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule FRET and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with 10 mis-matches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (e.g., transcription, replication, etc) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.
Paudel BP, Fiorini E, Börner R, et al., 2018, Optimal molecular crowding accelerates group II intron folding and maximizes catalysis, Proceedings of the National Academy of Sciences, Vol: 115, Pages: 11917-11922, ISSN: 0027-8424
Unlike in vivo conditions, group II intron ribozymes are known to require high magnesium(II) concentrations ([Mg2+]) and high temperatures (42 °C) for folding and catalysis in vitro. A possible explanation for this difference is the highly crowded cellular environment, which can be mimicked in vitro by macromolecular crowding agents. Here, we combined bulk activity assays and single-molecule Förster Resonance Energy Transfer (smFRET) to study the influence of polyethylene glycol (PEG) on catalysis and folding of the ribozyme. Our activity studies reveal that PEG reduces the [Mg2+] required, and we found an “optimum” [PEG] that yields maximum activity. smFRET experiments show that the most compact state population, the putative active state, increases with increasing [PEG]. Dynamic transitions between folded states also increase. Therefore, this study shows that optimal molecular crowding concentrations help the ribozyme not only to reach the native fold but also to increase its in vitro activity to approach that in physiological conditions.
Gahlon HL, Walker AR, Cisneros GA, et al., 2018, Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant, Physical Chemistry Chemical Physics, Vol: 20, Pages: 26892-26902, ISSN: 1463-9076
DNA synthesis, carried out by DNA polymerases, requires balancing speed and accuracy for faithful replication of the genome. High fidelity DNA polymerases contain a 3′–5′ exonuclease domain that can remove misincorporated nucleotides on the 3′ end of the primer strand, a process called proofreading. The E. coli replicative polymerase, DNA polymerase III, has spatially separated (∼55 Å apart) polymerase and exonuclease subunits. Here, we report on the dynamics of E. coli DNA polymerase III proofreading in the presence of its processivity factor, the β2-sliding clamp, at varying base pair termini using single-molecule FRET. We find that the binding kinetics do not depend on the base identity at the termini, indicating a tolerance for DNA mismatches. Further, our single-molecule data and MD simulations show two previously unobserved features: (1) DNA Polymerase III is a highly dynamic protein that adopts multiple conformational states while bound to DNA with matched or mismatched ends, and (2) an exonuclease-deficient DNA polymerase III has reduced conformational flexibility. Overall, our single-molecule experiments provide high time-resolution insight into a mechanism that ensures high fidelity DNA replication to maintain genome integrity.
Willhoft O, Ghoneim M, Lin C-L, et al., 2018, Structure and dynamics of the yeast SWR1:nucleosome complex, Science, Vol: 362, ISSN: 0036-8075
INTRODUCTIONCanonical nucleosomes contain two copies of each of four histone proteins: H2A, H2B, H3, and H4. However, variants of these histones can be inserted by adenosine triphosphate (ATP)–dependent chromatin-remodeling machines. The yeast SWR1 chromatin-remodeling complex, a member of the INO80 remodeler family, catalyzes the exchange of H2A-H2B dimers for dimers containing Htz1 (H2A.Z in human) in an ATP-dependent manner. However, the mechanism by which SWR1 exchanges histones is poorly understood. Despite having a DNA translocase subunit similar to that in the INO80 complex that slides nucleosomes, no net translocation of nucleosomes has been reported for SWR1. Consequently, the function of the ATPase activity, which is required for histone exchange in SWR1, has remained enigmatic.RATIONALETo obtain sufficient quantities for structural analysis, we generated the complete 14-subunit yeast SWR1 complex in insect cells. Binding of nucleosomes to SWR1 is stabilized in the presence of an ATP analog (ADP•BeF3), which we used to prepare a complex with a canonical yeast H2A-containing nucleosome. Structural analysis was undertaken by cryo–electron microscopy (cryo-EM). We also used single-molecule FRET (smFRET) techniques to probe the dynamics of nucleosomes bound to SWR1. Fluorescent probes were positioned on the H2A histones and the end of the DNA to monitor changes in nucleosome dynamics upon binding of SWR1 and ATP (or ATP analogs).RESULTSWe determined the cryo-EM structure of the SWR1-nucleosome complex at 3.6-Å resolution. The architecture of the complex shows how the SWR1 complex is assembled around a heterohexameric core of the RuvBL1 and RuvBL2 subunits. The Swr1 motor subunit binds at superhelical location 2 (SHL2), a position it shares in common with other remodelers but not with its most closely related complex, INO80, which binds at SHL6-SHL7. Binding of ATP or ADP•BeF3 to the SWR1-nucleosome complex induces substantial unwrap
Vamosi G, Rueda D, 2018, DNA Bends the Knee to Transcription Factors, BIOPHYSICAL JOURNAL, Vol: 114, Pages: 2253-2254, ISSN: 0006-3495
Ayala R, Willhoft O, Aramayo R, et al., 2018, Structure and regulation of the human INO80–nucleosome complex, Nature, Vol: 556, Pages: 391-395, ISSN: 0028-0836
Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines1. Biochemical studies2-4 have placed the motor domains of several remodellers on the superhelical location (SHL) 2 region of the nucleosome. Structural studies on Chd1 and Snf2 (RSC) in complex with nucleosomes5-7 have provided insights into the basic mechanism of nucleosome sliding by these complexes. However, how larger, multi-subunit remodelling complexes, such as INO80, interact with nucleosomes or how remodellers carry out functions such as nucleosome sliding8, histone exchange9, and nucleosome spacing10-12 remains poorly understood. Although some remodellers work as monomers13, others work as highly cooperative dimers11,14,15. Here we present the structure of the INO80 chromatin remodeller with a bound nucleosome revealing that INO80 interacts with nucleosomes in a unique manner with the motor domains located at the entry point to the wrap around the histone core rather than at SHL2. The Arp5-Ies6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This unique arrangement allows the H3 tails of the nucleosome to play a role in regulation, differing from other characterised remodellers.
Rudan M, Dib PB, Musa M, et al., 2018, Normal mitochondrial function in Saccharomyces cerevisiae has become dependent on inefficient splicing, eLife, Vol: 7, ISSN: 2050-084X
Self-splicing introns are mobile elements that have invaded a number of highlyconserved genes in prokaryotic and organellar genomes. Here, we show that deletion of theseselfish elements from the Saccharomyces cerevisiae mitochondrial genome is stressful to the host.A strain without mitochondrial introns displays hallmarks of the retrograde response, with alteredmitochondrial morphology, gene expression and metabolism impacting growth and lifespan.Deletion of the complete suite of mitochondrial introns is phenocopied by overexpression of thesplicing factor Mss116. We show that, in both cases, abnormally efficient transcript maturationresults in excess levels of mature cob and cox1 host mRNA. Thus, inefficient splicing has becomean integral part of normal mitochondrial gene expression. We propose that the persistence of S.cerevisiae self-splicing introns has been facilitated by an evolutionary lock-in event, where the hostgenome adapted to primordial invasion in a way that incidentally rendered subsequent intron lossdeleterious.
Walker A, Gahlon H, Rueda D, et al., 2018, Effects of a single point mutation and mismatched base on DNA polymerase III holoenzyme proofreading, 255th National Meeting and Exposition of the American-Chemical-Society (ACS) - Nexus of Food, Energy, and Water, Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
Autour A, Jeng S, Cawte A, et al., 2018, Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells, Nature Communications, Vol: 9, ISSN: 2041-1723
Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.
Cawte A, Jeng S, Autour A, et al., 2018, Cellular Imaging of Small RNAs using Fluorescent RNA-Mango Aptamers, 62nd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 215A-216A, ISSN: 0006-3495
Ghoneim M, Lin C-L, McCormack EA, et al., 2018, Dynamics of Eukaryotic Histone Exchange with Single Molecule Resolution, 62nd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 446A-446A, ISSN: 0006-3495
Liyanage PS, Walker AR, Brenlla A, et al., 2017, Bulky Lesion Bypass Requires Dpo4 Binding in Distinct Conformations., Sci Rep, Vol: 7
Translesion DNA synthesis is an essential process that helps resume DNA replication at forks stalled near bulky adducts on the DNA. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) that can be metabolically activated to benzo[a]pyrene diol epoxide (BPDE), which then can react with DNA to form carcinogenic DNA adducts. Here, we have used single-molecule florescence resonance energy transfer (smFRET) experiments, classical molecular dynamics simulations, and nucleotide incorporation assays to investigate the mechanism by which the model Y-family polymerase, Dpo4, bypasses a (+)-cis-B[a]P-N 2-dG adduct in DNA. Our data show that when (+)-cis-B[a]P-N 2-dG is the templating base, the B[a]P moiety is in a non-solvent exposed conformation stacked within the DNA helix, where it effectively blocks nucleotide incorporation across the adduct by Dpo4. However, when the media contains a small amount of dimethyl sulfoxide (DMSO), the adduct is able to move to a solvent-exposed conformation, which enables error-prone DNA replication past the adduct. When the primer terminates across from the adduct position, the addition of DMSO leads to the formation of an insertion complex capable of accurate nucleotide incorporation.
Zhang X, Aramayo RJ, Willhoft O, et al., 2017, CryoEM structures of the human INO80 chromatin remodelling complex, Nature Structural and Molecular Biology, Vol: 25, Pages: 37-44, ISSN: 1545-9985
Access to chromatin for processes such as DNA repair and transcription requires the sliding of nucleosomes along DNA. The multi-subunit INO80 chromatin remodelling complex has a particular role in DNA repair. Here we present the cryo electron microscopy structures of the active core complex of human INO80 at 9.6 Å with portions at 4.1 Å resolution along with reconstructions of combinations of subunits. Together these structures reveal the architecture of the INO80 complex, including Ino80 and actin-related proteins, which is assembled around a single Tip49a (RUVBL1) and Tip49b (RUVBL2) AAA+ heterohexamer. An unusual spoked-wheel structural domain of the Ino80 subunit is engulfed by this heterohexamer and the intimate association of this Ino80 domain with the heterohexamer is at the core of the complex. We also identify a cleft in RUVBL1 and RUVBL2, which forms a major interaction site for partner proteins and likely communicates partner-interactions with its nucleotide binding sites.
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