Analysis of the mechanism of DNA methylation involving DNMT1 and UHRF1 by using novel fluorescence tools – fluometadn

To reach our objectives, fluorescence spectroscopy appears as the technique of choice, due to its exquisite sensitivity (which allows working at low concentrations in solution) and its ability to site-specifically monitor conformational changes at the two partners binding site. In our case, the great challenge relies on the need to monitor these modifications at the DNA level, which implies to label it by a fluorescent reporter. This objective is very difficult to reach, as no fluorescent reporter, able to replace without any perturbation a natural base and keeping at the same time strong emissive properties and high sensitivity to the modifications of its environment, existed up to now. Thus, one of the major objectives was to synthesize such reporters, based on a family of fluorophores presenting a dual (two bands) emission spectrum, highly sensitive to the environment. By incorporating these reporters in double-stranded DNAs at different positions in respect to the UHRF1 binding site that includes the methylated base, we have characterized the binding of UHRF1 to these labeled sequences by fluorescence spectroscopy as well as by the stopped-flow fluorescence technique that allows monitoring the kinetics of interaction after rapid mixing of the two partners.

We synthesized new fluorescent analogs of nucleosides and demonstrated their high environment sensitivity. Sequences incorporating in different positions one of these analogs were synthesized in order to study the first step of the DNA methylation mechanism. For two of these positions, the reporter was found to only minimally perturb the duplex conformation and the binding of the protein. Fluorescence studies allowed us to monitor the binding kinetics of the SRA domain of UHRF1 as well as the flipping of the methylated base. The results were further confirmed using SRA mutants. Our data provided a better understanding of the recruitment mechanism of DNMT1 by UHRF1 as well as some clues for the development of UHRF1 inhibitors. Finally, this work allowed initiating new collaborations with international partners: Y. Tor (USA) and O. Fedorova (Russia) in order to solve new questions on UHRF1 and to find new applications for the present fluorescent reporters.

Two major achievements could be underlined. The first one is the synthesis of a series of fluorescent nucleoside surrogates for efficient characterization and quantification of protein/nucleic acid interactions using the multi-parametric and environment sensitivity properties of the reporting group. The second one is the fine description of the UHRF1 binding and base flipping mechanisms. Both have an important scientific impact. The fluorescent nucleoside surrogates could be used for answering a large number of questions in the very general field of protein/nucleic acid interactions. They have already been applied for investigating the kinetic mechanism of Endonuclease VIII and the nucleic acid chaperone properties of the nucleocapsid protein of HIV-1. The understanding of the initial steps of the replication of the DNA methylation pattern is important both on a fundamental and applied point of view. Indeed, we obtained key information on the base flipping process but also on the possible recruitment of DNMT1 by UHRF1. It will be possible now, to check this recruitment mechanism with our tools as well as the cooperation between the two proteins during the replication of the DNA methylation pattern. Moreover, both the UHRF-1 mediated base flipping and DNMT1 recruitment are obvious targets for designing new strategies for preventing the altered methylation profiles in several cancers.

The present work gave rise to thirteen published papers and seven papers either submitted or in preparation. Most of these papers concern the development and the characterization of new fluorescent nucleoside surrogates. Two papers deal with the interaction of UHRF1with hemi-methylated DNAs labeled by a fluorescent reporter. Two PhD theses were also dedicated to this work, one in chemistry (Nicolas Barthès, Nice University) and the second in biophysics (Vasyl Kilin, Strasbourg University).

Many fundamental cellular events such as replication, translation, transcription, DNA repair, DNA methylation and gene silencing involve complex machineries of nucleic acids and proteins. Due to the capability of fluorescence-based techniques to decrease the measurements down to a single molecule and due to the exquisite sensitivity of many fluorophores to their environment, fluorescence spectroscopy is a technique of choice to unravel the molecular mechanisms and dynamics of these machineries. However, this method is limited by the availability of optimized fluorophores that sensitively, site-specifically and multiparametrically monitor the conformational changes of nucleic acids during interaction with their protein partners. In this context, we propose to develop new chemical tools based on environmental sensitive fluorescent dyes of the 3-hydroxychrome (3HC) family, to study with state-of-the-art fluorescence techniques the dynamics and mechanism of DNA methylation by the UHRF1/DNMT1 protein tandem, which plays a key role in DNA replication by transferring the methylation pattern from the parent DNA strand to the newly synthesized strand. Moreover, as a consequence of its key role in epigenetics, DNMT1 has been recognized as an ideal target to fight cancers, since the latter are frequently characterized by an alteration of the DNA methylation pattern. Therefore, unravelling the successive molecular steps involved in the DNA methylation pattern inheritance is expected i) to reveal new molecular mechanistic aspects that could be potentially targeted for cancer therapy and ii) to suggest new assays that could be used to screen anticancer drugs. A first generation of nucleoside analogues based on 3HC was already synthesized and found to behave as a universal base that induces only limited destabilization in DNA and responds by a change of the ratio of its two emission bands to the binding of a nucleic acid binding protein, giving a first proof of concept of our approach. This interdisciplinary project at the interface between chemistry and biophysics will be performed by two highly complementary partners. Partner 1 is an expert in fluorescence spectroscopy and in 3HC probes as well as in protein/nucleic acid interaction and UHRF1. Therefore, partner 1 will select the appropriate 3HC chromophores for the development of the nucleoside analogues, characterize the photophysical properties of the synthesized nucleosides and the labelled oligonucleotides, and unravel the molecular steps involved in the methylation of the labelled oligonucleotides by the UHRF1/DNMT1 couple. Partner 2 is specialized in organic and medicinal chemistry, the design of fluorescent probes and nucleic acid chemistry. Partner 2 will thus design and synthesize the new fluorescent tools of this project, and include them in oligonucleotides (ODNs) by solid phase chemistry.