title: Dynamics of Methylated Cytosine Flipping by UHRF1.
abstract: 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.
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie , 74 Route du Rhin, 67401 Illkirch, France.
authors.forename: Vasyl
authors.initials: V
authors.lastname: Kilin
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie , 74 Route du Rhin, 67401 Illkirch, France.
authors.forename: Krishna
authors.initials: K
authors.lastname: Gavvala
authors.affiliation: Institut de Chimie de Nice, UMR 7272 CNRS, Université Côte d'Azur , Parc Valrose, 06108 Nice Cedex 2, France.
authors.forename: Nicolas P F
authors.initials: NP
authors.lastname: Barthes
authors.affiliation: Institut de Chimie de Nice, UMR 7272 CNRS, Université Côte d'Azur , Parc Valrose, 06108 Nice Cedex 2, France.
authors.forename: Benoît Y
authors.initials: BY
authors.lastname: Michel
authors.affiliation: TriLink BioTechnologies, LLC. , San Diego, California 92121, United States.
authors.forename: Dongwon
authors.initials: D
authors.lastname: Shin
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie , 74 Route du Rhin, 67401 Illkirch, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Boudier
authors.affiliation: LBPA, UMR 8113 CNRS, ENS Paris-Saclay, Université Paris Saclay , 94235 Cachan Cedex, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Mauffret
authors.affiliation: Department of Physics, Kiev National Taras Shevchenko University , Kiev 01601, Ukraine.
authors.forename: Valeriy
authors.initials: V
authors.lastname: Yashchuk
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie , 74 Route du Rhin, 67401 Illkirch, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Mousli
authors.affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964 CNRS UMR 7104, Université de Strasbourg , Illkirch 67000, France.
authors.forename: Marc
authors.initials: M
authors.lastname: Ruff
authors.affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964 CNRS UMR 7104, Université de Strasbourg , Illkirch 67000, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Granger
authors.affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964 CNRS UMR 7104, Université de Strasbourg , Illkirch 67000, France.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Eiler
authors.affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U964 CNRS UMR 7104, Université de Strasbourg , Illkirch 67000, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Bronner
authors.affiliation: Department of Chemistry and Biochemistry, University of California , San Diego, La Jolla, California 92093-0358, United States.
authors.forename: Yitzhak
authors.initials: Y
authors.lastname: Tor
authors.affiliation: Institut de Chimie de Nice, UMR 7272 CNRS, Université Côte d'Azur , Parc Valrose, 06108 Nice Cedex 2, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Burger
authors.affiliation: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de pharmacie , 74 Route du Rhin, 67401 Illkirch, France.
authors.forename: Yves
authors.identifier: 0000-0001-7328-8269
authors.initials: Y
authors.lastname: Mély
mesh_terms: D022762:CCAAT-Enhancer-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D003596:Cytosine / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D004261:DNA Replication; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D013816:Thermodynamics; D044767:Ubiquitin-Protein Ligases
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D003596:Cytosine; D004247:DNA; C406364:UHRF1 protein, human; D044767:Ubiquitin-Protein Ligases