127 results found
Aristodemou AEN, Rueda DS, Taylor GP, et al., 2023, The transcriptome of HTLV-1-infected primary cells following reactivation reveals changes to host gene expression central to the proviral life cycle
<jats:title>Abstract</jats:title><jats:p>Infections by Human T cell Leukaemia Virus type 1 (HTLV-1) persist for the lifetime of the host by integrating into the genome of CD4<jats:sup>+</jats:sup>T cells. Proviral gene expression is core to proviral survival and the maintenance of the proviral load, through the pro-proliferative changes it induces in infected cells. Despite their role in HTLV-1 infection and a persistent cytotoxic T lymphocyte response raised against them, proviral transcripts from the sense-strand are rarely detected in fresh cells extracted from the peripheral blood, and have recently been found to be expressed intermittently by a small subset of cells at a given time.<jats:italic>Ex vivo</jats:italic>culture of infected cells prompts synchronised proviral expression in infected cells from peripheral blood, allowing the study of factors involved in reactivation in primary cells. Here, we used bulk RNA-seq to examine the host transcriptome over six days<jats:italic>in vitro</jats:italic>, following proviral reactivation in primary peripheral CD4<jats:sup>+</jats:sup>T cells isolated from subjects with non-malignant HTLV-1 infection. Infected cells displayed a conserved response to reactivation, characterised by discrete stages of gene expression, cell division and subsequently horizontal transmission of the virus. We observed widespread changes in Polycomb gene expression following reactivation, including an increase in PRC2 transcript levels and diverse changes in the expression of PRC1 components. We hypothesize that these transcriptional changes constitute a negative feedback loop that maintains proviral latency by re-deposition of H2AK119ub1 following the end of proviral expression. Using RNAi, we found that certain deubiquitinases,<jats:italic>BAP1</jats:italic>,<jats:italic>USP14</jats:italic>and<jats:italic>OTUD5</jats:italic>each promote pr
Belan O, Sebald M, Adamowicz M, et al., 2022, POLQ seals post-replicative ssDNA gaps to maintain genome stability in BRCA-deficient cancer cells., Mol Cell, Vol: 82, Pages: 4664-4680.e9
POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability.
Moore G, Han Z, Xu J, et al., 2022, Dynamic Backtracking Regulates Lesion Bypass by RNAPII
<jats:title>Abstract</jats:title> <jats:p>The eukaryotic genome is prone to a high amount of DNA damage from intrinsic and extrinsic sources, causing transcriptional stress, including pausing, backtracking and stalling. If not rectified in time, these damages can further lead to transcriptional arrest and genome instability. Here, we develop a single-molecule FRET based elongation complex which allows us to insert various types of DNA damage into the transcribed region and study the effect they have on the dynamics of RNAPII transcription. We show that different DNA lesions cause a heterogenous effect on RNAPII. In some instances, such as oxidative lesions, RNAPII exhibits a high level of dynamic behaviour often backtracking up to 10 nt. While other damages, such as cyclo-butane pyrimidine dimers and abasic sites, can cause more significant static stalling. Furthermore, the repair factor Rad26 binds to RNAPII and alters these dynamics by pushing RNAPII further over the damage site and preventing long-range backtracking events.</jats:p>
Kaczmarczyk AP, Déclais A-C, Newton MD, et al., 2022, Search and processing of Holliday junctions within long DNA by junction-resolving enzymes, Nature Communications, Vol: 13, Pages: 1-13
Resolution of Holliday junctions is a critical intermediate step of homologous recombination in which junctions are processed by junction-resolving endonucleases. Although binding and cleavage are well understood, the question remains how the enzymes locate their substrate within long duplex DNA. Here we track fluorescent dimers of endonuclease I on DNA, presenting the complete single-molecule reaction trajectory for a junction-resolving enzyme finding and cleaving a Holliday junction. We show that the enzyme binds remotely to dsDNA and then undergoes 1D diffusion. Upon encountering a four-way junction, a catalytically-impaired mutant remains bound at that point. An active enzyme, however, cleaves the junction after a few seconds. Quantitative analysis provides a comprehensive description of the facilitated diffusion mechanism. We show that the eukaryotic junction-resolving enzyme GEN1 also undergoes facilitated diffusion on dsDNA until it becomes located at a junction, so that the general resolution trajectory is probably applicable to many junction resolving enzymes.
Kaczmarczyk A, Déclais A-C, Newton M, et al., 2022, Search and Processing of Holliday Junctions within Long DNA by Junction-Resolving Enzymes, Publisher: Research Square Platform LLC
<jats:title>Abstract</jats:title> <jats:p>Resolution of four-way Holliday junctions is a critical intermediate step of homologous recombination. DNA junctions must be processed by abundant junction-resolving endonucleases to cleave the covalent link between two chromosomes. Although the catalytic activity and interaction mode of endonucleases with Holliday junction have been characterized in great detail by biochemical and structural studies, it remains unclear how the enzymes find their substrate located within kilobase pairs of duplex DNA. Here, we present a complete single-molecule reaction trajectory for a junction-resolving enzyme finding and cleaving a Holliday junction. We employed correlative optical tweezers with confocal fluorescence microscopy to track a single dimer of endonuclease I on the DNA template in real-time. We observed that the enzyme binds remotely to the dsDNA and then undergoes 1D diffusion. Upon encountering the four-way junction, a catalytically impaired endonuclease I mutant remains bound at that point for long periods. An active enzyme, however, cleaves the junction after a few seconds. When an N-terminal truncated endonuclease mutant is used, the enzyme fails to diffuse along dsDNA and dissociates after short periods of time, indicating that DNA encirclement by the disordered N-terminus is a key requirement for 1D diffusion and efficient target localisation. Quantitative analysis of each of these stages revealed a comprehensive description of the facilitated diffusion mechanism. We show that the eukaryotic junction-resolving enzyme GEN1 also undergoes facilitated diffusion on dsDNA until it becomes located at a junction, so that the general resolution trajectory is likely to be applicable to most junction resolving enzymes.</jats:p>
Newton MD, Taylor BJ, Cuomo ME, et al., 2022, CRISPR/Cas9 On- and Off-Target Activity Using Correlative Force and Fluorescence Single-Molecule Microscopy., Methods Mol Biol, Vol: 2478, Pages: 349-378
The discovery of CRISPR/Cas9 as an easily programmable endonuclease heralds a new era of genetic manipulation. With this comes the prospect of novel gene therapy approaches, and the potential to cure previously untreatable genetic diseases. However, reports of spurious off-target editing by CRISPR/Cas9 pose a significant hurdle to realizing this potential. A deeper understanding of the factors that affect Cas9 specificity is vital for development of safe and efficient therapeutics. Here, we describe methods for the use of optical tweezers combined with confocal fluorescence microscopy and microfluidics for the analysis of on- and off-target activity of Cas9 activity.
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: 268-273, ISSN: 0028-0836
DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2,3,4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, 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 sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.
Rueda D, Cawte AD, Ino H, et al., 2021, Single-molecule RNA Imaging Using Mango II Arrays, Methods in Molecular Biology, Publisher: Humana Press
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.
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.
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
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
Boulton S, Anand R, Buechelmaier E, et al., 2021, HELQ is a dual function DSB repair enzyme modulated by RPA and RAD51, Publisher: Research Square Platform LLC
<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>
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
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.
Newton MD, Taylor BJ, Driessen RPC, et al., 2019, DNA stretching induces Cas9 off-target activity, Nature Structural and Molecular Biology, Vol: 26, Pages: 185-192, ISSN: 1545-9985
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.
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>
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
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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.
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