Publications
133 results found
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
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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.
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
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
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.
Lane DA, Lynch CJ, Millar C, et al., 2017, A common mechanism by which type 2A von Willebrand Disease mutations enhance ADAMTS13 proteolysis revealed with a von Willebrand Factor A2 domain FRET construct, PLoS ONE, Vol: 12, ISSN: 1932-6203
Rheological forces in the blood trigger the unfolding of von Willebrand factor (VWF) and its A2 domain, exposing the scissile bond for proteolysis by ADAMTS13. Under quiescent conditions, the scissile bond is hidden by the folded structure due to the stabilisation provided by the structural specialisations of the VWF A2 domain, a vicinal disulphide bond, a calcium binding site and a N1574-glycan.The reduced circulating high MW multimers of VWF in patients with type 2A von Willebrand disease (VWD) may be associated with mutations within the VWF A2 domain and this is attributed to enhanced ADAMTS13 proteolysis. We investigated 11 VWF A2 domain variants identified in patients with type 2A VWD. In recombinant full-length VWF, enhanced ADAMTS13 proteolysis was detected for all of the expressed variants in the presence of urea-induced denaturation. A subset of the FLVWF variants displayed enhanced proteolysis in the absence of urea. The mechanism of enhancement was investigated using a novel VWF A2 domain FRET construct. In the absence of induced unfolding, 7/8 of the expressed mutants exhibited a disrupted domain fold, causing spatial separation of the N- and C- termini. Three of the type 2A mutants were not secreted when studied within the VWF A2 domain FRET construct. Urea denaturation revealed for all 8 secreted mutants reduced unfolding cooperativity and stability of the VWF A2 domain. As folding stability was progressively disrupted, proteolysis by ADAMTS13 increased. Due to the range of folding stabilities and wide distribution of VWF A2 domain mutations studied, we conclude that these mutations disrupt regulated folding of the VWF A2 domain. They enhance unfolding by inducing separation of N- and C-termini, thereby promoting a more open conformation that reveals its binding sites for ADAMTS13 and the scissile bond.
Gahlon HL, Poudevigne-Durance P, Rueda D, 2017, Absence of nuclease activity in commonly used oxygen-scavenging systems, BMC Research Notes, Vol: 10, ISSN: 1756-0500
ObjectiveOxygen scavenging systems are routinely used during single-molecule imaging experiments to improve fluorescent dye stability. Previous work has shown nuclease contamination in the commonly used oxygen scavenging systems. This study evaluates the potential for nuclease contamination in these oxygen scavenging systems.ResultsLinear and plasmid DNA was incubated with two different oxygen scavenging systems (1) protocatechuic acid (PCA)-protocatechuate-3,4-dioxygenase (PCD) and (2) glucose-coupled glucose oxidase/catalase (GODCAT). No nucleic acid degradation was observed on single and double-stranded linear DNA and plasmid DNA, indicating the absence of nuclease contamination in these oxygen scavenging systems.
Rueda FO, Bista M, Newton MD, et al., 2017, Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease, Nature Communications, Vol: 8, ISSN: 2041-1723
The CRISPR–Cas9 RNA-guided endonuclease system allows precise and efficient modification of complex genomes and is continuously developed to enhance specificity, alter targeting and add new functional moieties. However, one area yet to be explored is the base chemistry of the associated RNA molecules. Here we show the design and optimisation of hybrid DNA–RNA CRISPR and tracr molecules based on structure-guided approaches. Through careful mapping of the ribose requirements of Cas9, we develop hybrid versions possessing minimal RNA residues, which are sufficient to direct specific nuclease activity in vitro and in vivo with reduced off-target activity. We identify critical regions within these molecules that require ribose nucleotides and show a direct correlation between binding affinity/stability and cellular activity. This is the first demonstration of a non-RNA-guided Cas9 endonuclease and first step towards eliminating the ribose dependency of Cas9 to develop a XNA-programmable endonuclease.
Gahlon HL, Romano LJ, Rueda D, 2017, Influence of DNA Lesions on Polymerase-Mediated DNA Replication at Single-Molecule Resolution., Chemical Research in Toxicology, Vol: 30, Pages: 1972-1983, ISSN: 0893-228X
Faithful replication of DNA is a critical aspect in maintaining genome integrity. DNA polymerases are responsible for replicating DNA, and high-fidelity polymerases do this rapidly and at low error rates. Upon exposure to exogenous or endogenous substances, DNA can become damaged and this can alter the speed and fidelity of a DNA polymerase. In this instance, DNA polymerases are confronted with an obstacle that can result in genomic instability during replication, for example, by nucleotide misinsertion or replication fork collapse. It is important to know how DNA polymerases respond to damaged DNA substrates to understand the mechanism of mutagenesis and chemical carcinogenesis. Single-molecule techniques have helped to improve our current understanding of DNA polymerase-mediated DNA replication, as they enable the dissection of mechanistic details that can otherwise be lost in ensemble-averaged experiments. These techniques have also been used to gain a deeper understanding of how single DNA polymerases behave at the site of the damage in a DNA substrate. In this review, we evaluate single-molecule studies that have examined the interaction between DNA polymerases and damaged sites on a DNA template.
Billman MR, Rueda D, Bangham CRM, 2017, Single-cell heterogeneity and cell-cycle-related viral gene bursts in the human leukaemia virus HTLV-1 [version 1; peer review: 2 approved, 1 approved with reservations], Wellcome Open Research, Vol: 2, Pages: 87-87, ISSN: 2398-502X
Background: The human leukaemia virus HTLV-1 expresses essential accessory genes that manipulate the expression, splicing and transport of viral mRNAs. Two of these genes, tax and hbz, also promote proliferation of the infected cell, and both genes are thought to contribute to oncogenesis in adult T-cell leukaemia/lymphoma. The regulation of HTLV-1 proviral latency is not understood. tax, on the proviral plus strand, is usually silent in freshly-isolated cells, whereas the minus-strand-encoded hbz gene is persistently expressed at a low level. However, the persistently activated host immune response to Tax indicates frequent expression of tax in vivo. Methods: We used single-molecule RNA-FISH to quantify the expression of HTLV-1 transcripts at the single-cell level in a total of >19,000 cells from five T-cell clones, naturally infected with HTLV-1, isolated by limiting dilution from peripheral blood of HTLV-1-infected subjects. Results: We found strong heterogeneity both within and between clones in the expression of the proviral plus-strand (detected by hybridization to the tax gene) and the minus-strand ( hbz gene). Both genes are transcribed in bursts; tax expression is enhanced in the absence of hbz, while hbz expression increased in cells with high tax expression. Surprisingly, we found that hbz expression is strongly associated with the S and G 2/M phases of the cell cycle, independent of tax expression. Contrary to current belief, hbz is not expressed in all cells at all times, even within one clone. In hbz-positive cells, the abundance of hbz transcripts showed a very strong positive linear correlation with nuclear volume. Conclusions: The occurrence of intense, intermittent plus-strand gene bursts in independent primary HTLV-1-infected T-cell clones from unrelated individuals strongly suggests that the HTLV-1 plus-strand is expressed in bursts in vivo. Our results offer an explanat
Perez MEF, Bloznelyte K, Merkenschlager M, et al., 2017, Visualizing CTCF-mediated DNA looping at the single-molecule level, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S141-S141, ISSN: 0175-7571
Cawte A, Jeng S, Autour A, et al., 2017, Cellular imaging of small RNAs using fluorescent RNA-Mango based aptamers, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S292-S292, ISSN: 0175-7571
Newton MD, Driessen R, Taylor BJ, et al., 2017, Force induced off-target binding of CRISPR/Cas9 with single molecule resolution, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S146-S146, ISSN: 0175-7571
Gahlon HL, Zhao G, Fernandez-Leiro R, et al., 2017, Dynamic proofreading in bacterial DNA polymerase III, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S142-S142, ISSN: 0175-7571
Xie SQ, Rueda D, 2017, Imaging endogenous activation-induced cytidine deaminase (AID) regulation in mammalian B cells, 19th IUPAB Congress / 11th EBSA Congress, Publisher: SPRINGER, Pages: S371-S371, ISSN: 0175-7571
Wigley DB, Willhoft O, McCormack EA, et al., 2017, Cross-talk within a functional INO80 complex dimer regulates nucleosome sliding, eLife, Vol: 6, ISSN: 2050-084X
Several chromatin remodellers have the ability to space nucleosomes on DNA. For ISWI remodellers, this involves an interplay between H4 histone tails, the AutoN and NegC motifs of the motor domains that together regulate ATPase activity and sense the length of DNA flanking the nucleosome. By contrast, the INO80 complex also spaces nucleosomes but is not regulated by H4 tails and lacks the AutoN and NegC motifs. Instead nucleosome sliding requires cooperativity between two INO80 complexes that monitor DNA length simultaneously on either side of the nucleosome during sliding. The C-terminal domain of the human Ino80 subunit (Ino80CTD) binds cooperatively to DNA and dimerisation of these domains provides crosstalk between complexes. ATPase activity, rather than being regulated, instead gradually becomes uncoupled as nucleosome sliding reaches an end point and this is controlled by the Ino80CTD. A single active ATPase motor within the dimer is sufficient for sliding.
Barbieri M, Xie SQ, Triglia ET, et al., 2017, Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells, NATURE STRUCTURAL & MOLECULAR BIOLOGY, Vol: 24, Pages: 515-+, ISSN: 1545-9993
Paudel BP, Rueda D, 2017, Correction to “Molecular Crowding Accelerates Ribozyme Docking and Catalysis”, Journal of the American Chemical Society, Vol: 139, Pages: 4582-4582, ISSN: 0002-7863
Supporting Information, page S9. An error was present in Figure S6c in the original publication. The corrected Supporting Information is provided here.
Liang Z, Brown KE, Carroll T, et al., 2017, A high-resolution map of transcriptional repression, ELIFE, Vol: 6, ISSN: 2050-084X
Turning genes on and off is essential for development and homeostasis, yet little is known about the sequence and causal role of chromatin state changes during the repression of active genes. This is surprising, as defective gene silencing underlies developmental abnormalities and disease. Here we delineate the sequence and functional contribution of transcriptional repression mechanisms at high temporal resolution. Inducible entry of the NuRD-interacting transcriptional regulator Ikaros into mouse pre-B cell nuclei triggered immediate binding to target gene promoters. Rapid RNAP2 eviction, transcriptional shutdown, nucleosome invasion, and reduced transcriptional activator binding required chromatin remodeling by NuRD-associated Mi2beta/CHD4, but were independent of HDAC activity. Histone deacetylation occurred after transcriptional repression. Nevertheless, HDAC activity contributed to stable gene silencing. Hence, high resolution mapping of transcriptional repression reveals complex and interdependent mechanisms that underpin rapid transitions between transcriptional states, and elucidates the temporal order, functional role and mechanistic separation of NuRD-associated enzymatic activities.
Barbieri M, Xie SQ, Torlai Triglia E, et al., 2017, Active and poised promoter states drive folding of the extended<i>HoxB</i>locus in mouse embryonic stem cells
<jats:title>Abstract</jats:title><jats:p>Gene expression states influence the three-dimensional conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine<jats:italic>HoxB</jats:italic>locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features, which define promoter activation states or CTCF binding sites. Single cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fibre fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions.</jats:p>
Cawte A, Jeng S, Unrau P, et al., 2017, Live cell imaging of genomic loci using fluorescent RNA aptamers, 61st Annual Meeting of the Biophysical-Society, Publisher: Biophysical Society, Pages: 69A-69A, ISSN: 0006-3495
Perez MEF, Bloznelyte K, Merkenschlager M, et al., 2017, Visualizing CTCF mediated DNA looping at the single molecule level, 61st Annual Meeting of the Biophysical-Society, Publisher: Biophysical Society, Pages: 169A-170A, ISSN: 0006-3495
Gahlon HL, Zhao G, Fernandez-Leiro R, et al., 2017, Dynamic proofreading in the bacterial DNA polymerase, 58th Annual Meeting of the Biophysical-Society, Publisher: Biophysical Society, Pages: 513A-514A, ISSN: 0006-3495
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