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  • 1.
    Amselem, Elias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Kipper, Kalle
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Real- Time Single Protein Tracking with Polarization Readout using a Confocal Microscope2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 295A-295AArticle in journal (Other academic)
  • 2.
    Ballet, Caroline
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Correia, Mario S. P.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Conway, Louis P.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Locher, Theresa L.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Lehmann, Laura C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Garg, Neeraj
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vujasinovic, Miroslav
    Karolinska Univ Hosp, Dept Digest Dis, Stockholm, Sweden.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lohr, J. -Matthias
    Karolinska Inst, Dept Clin Sci Intervent & Technol CLINTEC, Stockholm, Sweden;Karolinska Univ Hosp, Dept Digest Dis, Stockholm, Sweden.
    Globisch, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery. Uppsala University, Science for Life Laboratory, SciLifeLab.
    New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites2018In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 9, no 29, p. 6233-6239Article in journal (Refereed)
    Abstract [en]

    Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.

  • 3.
    Bowman, Gregory D.
    et al.
    Johns Hopkins Univ, Thomas C Jenkins Dept Biophys, Baltimore, MD 21218 USA.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Remodeling the genome with DNA twists2019In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 366, no 6461, p. 35-36Article in journal (Other academic)
  • 4.
    Harada, Bryan T.
    et al.
    Harvard Univ, Grad Program Biophys, Cambridge, MA 02138 USA.;Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USA..
    Hwang, William L.
    Harvard Univ, Grad Program Biophys, Cambridge, MA 02138 USA.;Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USA.;Harvard Univ, Sch Med, Harvard MIT MD PhD Program, Boston, MA USA..
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USA.;Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA..
    Chatterjee, Nilanjana
    Univ Texas MD Anderson Canc Ctr, Dept Epigenet & Mol Carcinogenesis, Smithville, TX USA.;Univ Calif San Francisco, Sch Med, San Francisco, CA USA..
    Bartholomew, Blaine
    Univ Texas MD Anderson Canc Ctr, Dept Epigenet & Mol Carcinogenesis, Smithville, TX USA..
    Zhuang, Xiaowei
    Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USA.;Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA.;Harvard Univ, Dept Phys, Cambridge, MA 02138 USA..
    Stepwise nucleosome translocation by RSC remodeling complexes2016In: eLIFE, E-ISSN 2050-084X, Vol. 5, article id e10051Article in journal (Refereed)
    Abstract [en]

    The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1-2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome.

  • 5.
    Kipper, Kalle
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eremina, Nadja
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tubasum, Sumera
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mao, Guanzhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lehmann, Laura Christina
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 6, article id e0198416Article in journal (Refereed)
    Abstract [en]

    The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis- based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O-1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

  • 6.
    Lehmann, Laura C.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hewitt, Graeme
    Francis Crick Inst, 1 Midland Rd, London NW1 1AT, England..
    Aibara, Shintaro
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, S-17165 Solna, Sweden..
    Leitner, Alexander
    Swiss Fed Inst Technol, Inst Mol Syst Biol, Dept Biol, CH-8093 Zurich, Switzerland..
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Maslen, Sarah L.
    MRC Lab Mol Biol, Francis Crick Ave,Cambridge Biomed Campus, Cambridge CB2 0QH, England..
    Maturi, Varun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Chen, Yang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Skehel, J. Mark
    MRC Lab Mol Biol, Francis Crick Ave,Cambridge Biomed Campus, Cambridge CB2 0QH, England..
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Boulton, Simon J.
    Francis Crick Inst, 1 Midland Rd, London NW1 1AT, England..
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mechanistic Insights into Autoinhibition of the Oncogenic Chromatin Remodeler ALC12017In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 68, no 5, p. 847-859.e7Article in journal (Refereed)
    Abstract [en]

    Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying-mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.

  • 7.
    Levendosky, Robert F.
    et al.
    Johns Hopkins Univ, TC Jenkins Dept Biophys, Baltimore, MD 21218 USA..
    Sabantsev, Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bowman, Gregory D.
    Johns Hopkins Univ, TC Jenkins Dept Biophys, Baltimore, MD 21218 USA..
    The Chd1 chromatin remodeler shifts hexasomes unidirectionally2016In: eLIFE, E-ISSN 2050-084X, Vol. 5, article id e21356Article in journal (Refereed)
    Abstract [en]

    Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, providing a useful tool for interrogating chromatin enzymes and allowing for the generation of nucleosomes with precisely defined asymmetry. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo.

  • 8.
    Liao, Qinghua
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Lüking, Malin
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Krueger, Dennis M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. German Ctr Neurodegenerat Dis, Bioinformat Unit, Dept Epigenet & Syst Med Neurodegenerat Dis, Siebold Str 3A, D-37075 Gottingen, Germany.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kasson, Peter M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Long Time-Scale Atomistic Simulations of the Structure and Dynamics of Transcription Factor-DNA Recognition2019In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 123, no 17, p. 3576-3590Article in journal (Refereed)
    Abstract [en]

    Recent years have witnessed an explosion of interest in computational studies of DNA binding proteins, including both coarse grained and atomistic simulations of transcription factor-DNA recognition, to understand how these transcription factors recognize their binding sites on the DNA with such exquisite specificity. The present study performs microsecond time scale all-atom simulations of the dimeric form of the lactose repressor (Lad), both in the absence of any DNA and in the presence of both specific and nonspecific complexes, considering three different DNA sequences. We examine, specifically, the conformational differences between specific and nonspecific protein DNA interactions, as well as the behavior of the helix-turn-helix motif of Lad when interacting with the DNA. Our simulations suggest that stable Lad binding occurs primarily to bent A-form DNA, with a loss of Lad conformational entropy and optimization of correlated conformational equilibria across the protein. In addition, binding to the specific operator sequence involves a slightly larger number of stabilizing DNA protein hydrogen bonds (in comparison to nonspecific complexes), which may account for the experimentally observed specificity for this operator. In doing so, our simulations provide a detailed atomistic description of potential structural drivers for LacI selectivity.

  • 9.
    Marklund, Emil
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Amselem, Elias
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Kipper, Kalle
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Measuring the Orientation of Single Proteins Interacting with DNA using Fluorescence Polarization Microscopy2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 169A-169AArticle in journal (Other academic)
  • 10.
    Romilly, Cedric
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5 ' structure element2019In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 116, no 32, p. 15901-15906Article in journal (Refereed)
    Abstract [en]

    In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking-immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30 Delta S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5'-end stem-loop structure. Removal of the latter by 5'-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (similar to 100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.

  • 11.
    Sabantsev, Anton
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Levendosky, Robert F.
    Johns Hopkins Univ, TC Jenkins Dept Biophys, Baltimore, MD 21218 USA.
    Zhuang, Xiaowei
    Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USA;Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA;Harvard Univ, Dept Phys, Cambridge, MA 02138 USA.
    Bowman, Gregory D.
    Johns Hopkins Univ, TC Jenkins Dept Biophys, Baltimore, MD 21218 USA.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Direct observation of coordinated DNA movements on the nucleosome during chromatin remodelling2019In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 1720Article in journal (Refereed)
    Abstract [en]

    ATP-dependent chromatin remodelling enzymes (remodellers) regulate DNA accessibility in eukaryotic genomes. Many remodellers reposition (slide) nucleosomes, however, how DNA is propagated around the histone octamer during this process is unclear. Here we examine the real-time coordination of remodeller-induced DNA movements on both sides of the nucleosome using three-colour single-molecule FRET. During sliding by Chd1 and SNF2h remodellers, DNA is shifted discontinuously, with movement of entry-side DNA preceding that of exit-side DNA. The temporal delay between these movements implies a single ratelimiting step dependent on ATP binding and transient absorption or buffering of at least one base pair. High-resolution cross-linking experiments show that sliding can be achieved by buffering as few as 3 bp between entry and exit sides of the nucleosome. We propose that DNA buffering ensures nucleosome stability during ATP-dependent remodelling, and provides a means for communication between remodellers acting on opposite sides of the nucleosome.

  • 12.
    Saupe, Falk
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Reichel, Matthias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huijbers, Elisabeth J. M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Femel, Julia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Markgren, Per-Olof
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Andersson, C. Evalena
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Hellman, Lars T.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Olsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, Dept Med Biochem & Microbiol, Husargatan 3,Box 582, SE-75123 Uppsala, Sweden..
    Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously2017In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 31, no 3, p. 1204-1214Article in journal (Refereed)
    Abstract [en]

    With the aim to improve the efficacy of therapeutic vaccines that target self-antigens, we have developed a novel fusion protein vaccine on the basis of the C-terminal multimerizing end of the variable lymphocyte receptor B (VLRB), the Ig equivalent in jawless fishes. Recombinant vaccines were produced in Escherichia coli by fusing the VLRB sequence to 4 different cancer-associated target molecules. The anti-self-immune response generated in mice that were vaccinated with VLRB vaccines was compared with the response in mice that received vaccines that contained bacterial thioredoxin (TRX), previously identified as an efficient carrier. The anti-self-Abswere analyzed with respect to titers, binding properties, and duration of response. VLRB-vaccinatedmice displayed a 2-to 10-fold increase in anti-self-Ab titers and a substantial decrease in Abs against the foreign part of the fusion protein compared with the response in TRX-vaccinated mice (P < 0.01). VLRB-generated Ab response had duration similar to the corresponding TRX-generatedAbs, but displayed a higher diversity in binding characteristics. Of importance, VLRB vaccines could sustain an immune response against several targets simultaneously. VLRB vaccines fulfill several key criteria for an efficient therapeutic vaccine that targets self-antigens as a result of its small size, its multimerizing capacity, and nonexposed foreign sequences in the fusion protein.- Saupe, F., Reichel, M., Huijbers, E. J. M., Femel, J., Markgren, P.- O., Andersson, C. E., Deindl, S., Danielson, U. H., Hellman, L. T., Olsson, A.- K. Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

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