Dynamics of Methylated Cytosine Flipping by UHRF1
| Autor: | Marc Ruff, Benoît Y. Michel, Sylvia Eiler, Florence Granger, Vasyl Kilin, Olivier Mauffret, Nicolas Barthes, Yves Mély, Christian Bronner, Yitzhak Tor, Krishna Gavvala, Alain Burger, Valeriy M. Yashchuk, Dongwon Shin, Marc Mousli, Christian Boudier |
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| Přispěvatelé: | Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Group of Applied Physics - Biophotonics [Geneva] (GAP-Biophotonics), Group of Applied Physics [Geneva] (GAP), University of Geneva [Switzerland]-University of Geneva [Switzerland], Laboratoire de Bioimagerie et Pathologies (LBP), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Centre d’Ecologie Fonctionnelle et Evolutive (CEFE), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Université Paul-Valéry - Montpellier 3 (UPVM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut de Recherche pour le Développement (IRD [France-Sud]), Siemens Industry Software SAS [Roanne], Laboratoire de Biophotonique et Pharmacologie - UMR 7213 (LBP), Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA)), Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Cachan (ENS Cachan), Lawrence Berkeley National Laboratory [Berkeley] (LBNL), Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
DNA Replication
0301 basic medicine Stereochemistry Ubiquitin-Protein Ligases [SDV]Life Sciences [q-bio] Biochemistry DNA methyltransferase Fluorescence Article Catalysis Nucleobase Cytosine 03 medical and health sciences chemistry.chemical_compound Colloid and Surface Chemistry Genetics Humans ComputingMilieux_MISCELLANEOUS Molecular Structure DNA replication DNA General Chemistry DNA Methylation Kinetics 030104 developmental biology chemistry CpG site Chemical Sciences DNA methylation CCAAT-Enhancer-Binding Proteins Thermodynamics Nucleoside |
| Zdroj: | Journal of the American Chemical Society Journal of the American Chemical Society, American Chemical Society, 2017, 139 (6), pp.2520-2528. ⟨10.1021/jacs.7b00154⟩ Journal of the American Chemical Society, vol 139, iss 6 |
| ISSN: | 0002-7863 1520-5126 |
| DOI: | 10.1021/jacs.7b00154⟩ |
| Popis: | DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase. To accomplish this aim, we have utilized two distinct fluorescent nucleobase surrogates, 2-thienyl-3-hydroxychromone nucleoside (3HCnt) and thienoguanosine (thG), incorporated at different positions into hemimethylated (HM) and nonmethylated (NM) DNA duplexes. Large fluorescence changes were associated with mC flipping in HM duplexes, showing the outstanding sensitivity of both nucleobase surrogates to the small structural changes accompanying base flipping. Importantly, the nucleobase surrogates marginally affected the structure of the duplex and its affinity for SRA at positions where they were responsive to base flipping, illustrating their promise as nonperturbing probes for monitoring such events. Stopped-flow studies using these two distinct tools revealed the fast kinetics of SRA binding and sliding to NM duplexes, consistent with its reader role. In contrast, the kinetics of mC flipping was found to be much slower in HM duplexes, substantially increasing the lifetime of CpG-bound UHRF1, and thus the probability of recruiting DNMT1 to faithfully duplicate the DNA methylation profile. The fluorescence-based approach using these two different fluorescent nucleoside surrogates advances the mechanistic understanding of the UHRF1/DNMT1 tandem and the development of assays for the identification of base flipping inhibitors. |
| Databáze: | OpenAIRE |
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