Étude structurale et fonctionnelle de la fidélité des ADN polymérases X spécialisées dans la réparation des cassures doubles brins programmées chez Paramecium tetraurelia

The unicellular eukaryote Paramecium tetraurelia is a binucleate organism, which loses the nucleus required for gene expression (somatic) during reproduction. This nucleus must therefore be regenerated from its other nucleus (germinal), which is diploid. This regeneration involves numerous replications of the genome, but above all massive rearrangements, some of which consist in the programmed introduction of double-strand breaks at thousands of sites in the genome, in order to eliminate insertion sequences that break the reading frame in many genes. Once these breaks have been introduced, they are repaired by a system that relies on proteins involved in non-homologous repair, or NHEJ (Ku70/80, DNA-PKcs, Ligase IV, XRCC4) including 4 DNA polymerases. However, there is a major difference between classical NHEJ, which is known for its high error rate, and NHEJ in paramecium, which makes virtually no errors. The aim of this thesis is to explain the fidelity of this system, focusing on the DNA polymerases involved in this repair in Paramecium tetraurelia.Initially, a bioinformatics approach was used to hypothesize the reasons for the fidelity of these enzymes, by studying in depth the classification of DNA polymerases of family X. Following an enzymatic study of Paramecium tetraurelia DNA polymerases, which demonstrated their similarities to λ and β DNA polymerases, as well as their high fidelity, the existence of two mechanisms that could explain this fidelity was demonstrated. To this end, the enzymatic activity of DNA polymerase λ mutants was tested, and their structure obtained by X-ray crystallography. A first mechanism, similar to that encountered in DNA polymerase β, is based on local conformational changes within the enzyme's catalytic site. The second mechanism, uncharacterized until now, uses a 10-residue loop to stabilize the DNA within the active site, only in the presence of a correct nucleotide, and is found in DNA polymerase λ.These new insights into the molecular basis of X-family DNA polymerase fidelity provide a better understanding of Paramecium tetraurelia NHEJ fidelity, which may lead to a broader understanding of NHEJ and its implications in the immune system and carcinogenesis.L'eucaryote unicellulaire Paramecium tetraurelia est un organisme binucléé, qui a pour particularité de perdre lors de sa reproduction le noyau nécessaire à l'expression de ses gènes (somatique). Celui-ci doit donc être régénéré à partir de son autre noyau (germinal), quant à lui diploïde. Cette régénération passe par de nombreuses réplications du génome, mais surtout par des réarrangements massifs, dont certains consistent en l'introduction programmée de cassures double-brin à des milliers de sites dans le génome, afin d'éliminer des séquences d'insertion qui cassent le cadre de lecture dans de nombreux gènes. Une fois ces cassures introduites, elles sont réparées par un système qui repose sur des protéines impliquées dans la réparation non-homologue, ou NHEJ (Ku70/80, DNA-PKcs, Ligase IV, XRCC4) et sur 4 ADN polymérases. Cependant, il existe une différence majeure entre le NHEJ, qui est connu pour son fort taux d'erreurs, et le NHEJ chez la paramécie qui ne fait quasiment pas d'erreurs. L'objectif des travaux de cette thèse est d'expliquer la fidélité de ce système, en se focalisant sur les ADN polymérases impliquées dans cette réparation chez Paramecium tetraurelia.Dans un premier temps, une approche bio-informatique a été utilisée afin d'émettre des hypothèses sur les raisons de la fidélité de ces enzymes, en étudiant de façon approfondie la classification des ADN polymérases de la famille X. Après une étude enzymatique des ADN polymérases de Paramecium tetraurelia ayant permis de montrer leurs similitudes avec les ADN polymérases λ et β ainsi que leur grande fidélité, l'existence de deux mécanismes pouvant expliquer cette fidélité a été démontrée. Pour cela, l'activité enzymatique de mutants de l'ADN polymérase λ a été testée, et leur structure a été obtenue par cristallographie aux rayons X. Un premier mécanisme, similaire à celui rencontré chez l'ADN polymérase β, se base sur des changements conformationnels locaux au sein du site catalytique de l'enzyme. Le second mécanisme, jusqu'ici non caractérisé, utilise une boucle de 10 résidus pour stabiliser l'ADN au sein du site actif, uniquement en présence d'un nucléotide correct, et est retrouvé chez l'ADN polymérase λ. Ces nouvelles connaissances sur les bases moléculaires de la fidélité des ADN polymérases de la famille X apportent une meilleure compréhension de la fidélité du NHEJ de Paramecium tetraurelia, ce qui pourra permettre d'élargir les connaissances sur le NHEJ et ses implications dans le système immunitaire et dans la carcinogenèse

Étude structurale et fonctionnelle de la fidélité des ADN polymérases X spécialisées dans la réparation des cassures doubles brins programmées chez Paramecium tetraurelia

The unicellular eukaryote Paramecium tetraurelia is a binucleate organism, which loses the nucleus required for gene expression (somatic) during reproduction. This nucleus must therefore be regenerated from its other nucleus (germinal), which is diploid. This regeneration involves numerous replications of the genome, but above all massive rearrangements, some of which consist in the programmed introduction of double-strand breaks at thousands of sites in the genome, in order to eliminate insertion sequences that break the reading frame in many genes. Once these breaks have been introduced, they are repaired by a system that relies on proteins involved in non-homologous repair, or NHEJ (Ku70/80, DNA-PKcs, Ligase IV, XRCC4) including 4 DNA polymerases. However, there is a major difference between classical NHEJ, which is known for its high error rate, and NHEJ in paramecium, which makes virtually no errors. The aim of this thesis is to explain the fidelity of this system, focusing on the DNA polymerases involved in this repair in Paramecium tetraurelia.Initially, a bioinformatics approach was used to hypothesize the reasons for the fidelity of these enzymes, by studying in depth the classification of DNA polymerases of family X. Following an enzymatic study of Paramecium tetraurelia DNA polymerases, which demonstrated their similarities to λ and β DNA polymerases, as well as their high fidelity, the existence of two mechanisms that could explain this fidelity was demonstrated. To this end, the enzymatic activity of DNA polymerase λ mutants was tested, and their structure obtained by X-ray crystallography. A first mechanism, similar to that encountered in DNA polymerase β, is based on local conformational changes within the enzyme's catalytic site. The second mechanism, uncharacterized until now, uses a 10-residue loop to stabilize the DNA within the active site, only in the presence of a correct nucleotide, and is found in DNA polymerase λ.These new insights into the molecular basis of X-family DNA polymerase fidelity provide a better understanding of Paramecium tetraurelia NHEJ fidelity, which may lead to a broader understanding of NHEJ and its implications in the immune system and carcinogenesis.L'eucaryote unicellulaire Paramecium tetraurelia est un organisme binucléé, qui a pour particularité de perdre lors de sa reproduction le noyau nécessaire à l'expression de ses gènes (somatique). Celui-ci doit donc être régénéré à partir de son autre noyau (germinal), quant à lui diploïde. Cette régénération passe par de nombreuses réplications du génome, mais surtout par des réarrangements massifs, dont certains consistent en l'introduction programmée de cassures double-brin à des milliers de sites dans le génome, afin d'éliminer des séquences d'insertion qui cassent le cadre de lecture dans de nombreux gènes. Une fois ces cassures introduites, elles sont réparées par un système qui repose sur des protéines impliquées dans la réparation non-homologue, ou NHEJ (Ku70/80, DNA-PKcs, Ligase IV, XRCC4) et sur 4 ADN polymérases. Cependant, il existe une différence majeure entre le NHEJ, qui est connu pour son fort taux d'erreurs, et le NHEJ chez la paramécie qui ne fait quasiment pas d'erreurs. L'objectif des travaux de cette thèse est d'expliquer la fidélité de ce système, en se focalisant sur les ADN polymérases impliquées dans cette réparation chez Paramecium tetraurelia.Dans un premier temps, une approche bio-informatique a été utilisée afin d'émettre des hypothèses sur les raisons de la fidélité de ces enzymes, en étudiant de façon approfondie la classification des ADN polymérases de la famille X. Après une étude enzymatique des ADN polymérases de Paramecium tetraurelia ayant permis de montrer leurs similitudes avec les ADN polymérases λ et β ainsi que leur grande fidélité, l'existence de deux mécanismes pouvant expliquer cette fidélité a été démontrée. Pour cela, l'activité enzymatique de mutants de l'ADN polymérase λ a été testée, et leur structure a été obtenue par cristallographie aux rayons X. Un premier mécanisme, similaire à celui rencontré chez l'ADN polymérase β, se base sur des changements conformationnels locaux au sein du site catalytique de l'enzyme. Le second mécanisme, jusqu'ici non caractérisé, utilise une boucle de 10 résidus pour stabiliser l'ADN au sein du site actif, uniquement en présence d'un nucléotide correct, et est retrouvé chez l'ADN polymérase λ. Ces nouvelles connaissances sur les bases moléculaires de la fidélité des ADN polymérases de la famille X apportent une meilleure compréhension de la fidélité du NHEJ de Paramecium tetraurelia, ce qui pourra permettre d'élargir les connaissances sur le NHEJ et ses implications dans le système immunitaire et dans la carcinogenèse

Reclassification of family A DNA polymerases reveals novel functional subfamilies and distinctive structural features

International audienceFamily A DNA polymerases (PolAs) form an important and well-studied class of extant polymerases participating in DNA replication and repair. Nonetheless, despite the characterization of multiple subfamilies in independent, dedicated works, their comprehensive classification thus far is missing. We therefore re-examine all presently available PolA sequences, converting their pairwise similarities into positions in Euclidean space, separating them into 19 major clusters. While 11 of them correspond to known subfamilies, eight had not been characterized before. For every group, we compile their general characteristics, examine their phylogenetic relationships and perform conservation analysis in the essential sequence motifs. While most subfamilies are linked to a particular domain of life (including phages), one subfamily appears in Bacteria, Archaea and Eukaryota. We also show that two new bacterial subfamilies contain functional enzymes. We use AlphaFold2 to generate high-confidence prediction models for all clusters lacking an experimentally determined structure. We identify new, conserved features involving structural alterations, ordered insertions and an apparent structural incorporation of a uracil-DNA glycosylase (UDG) domain. Finally, genetic and structural analyses of a subset of T7-like phages indicate a splitting of the 3′–5′ exo and pol domains into two separate genes, observed in PolAs for the first time

Crystal structure of chloroplast fructose-1,6-bisphosphate aldolase from the green alga Chlamydomonas reinhardtii

Abstract The Calvin-Benson cycle fixes carbon dioxide into organic triosephosphates through the collective action of eleven conserved enzymes. Regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco-mediated carboxylation, requires two lyase reactions catalyzed by fructose-1,6-bisphosphate aldolase (FBA). While cytoplasmic FBA has been extensively studied in non-photosynthetic organisms, functional and structural details are limited for chloroplast FBA encoded by oxygenic phototrophs. Here we determined the crystal structure of plastidial FBA from the unicellular green alga Chlamydomonas reinhardtii (Cr). We confirm that CrFBA folds as a TIM barrel, describe its catalytic pocket and homo-tetrameric state. Multiple sequence profiling classified the photosynthetic paralogs of FBA in a distinct group from non-photosynthetic paralogs. We mapped the sites of thiol- and phospho-based post-translational modifications known from photosynthetic organisms and predict their effects on enzyme catalysis

Crystal structure of chloroplast fructose-1,6-bisphosphate aldolase from the green alga Chlamydomonas reinhardtii

International audienceResidue coevolution within and between proteins is used as a marker of physical interaction and/or residue functional cooperation. Pairs or groups of coevolving residues are extracted from multiple sequence alignments based on a variety of computational approaches. However, coevolution signals emerging in subsets of sequences might be lost if the full alignment is considered. iBIS2Analyzer is a web server dedicated to a phylogeny-driven coevolution analysis of protein families with different evolutionary pressure. It is based on the iterative version, iBIS2, of the coevolution analysis method BIS, Blocks in Sequences. iBIS2 is designed to iteratively select and analyse subtrees in phylogenetic trees, possibly large and comprising thousands of sequences. With iBIS2Analyzer, openly accessible at http://ibis2analyzer.lcqb.upmc.fr/, the user visualizes, compares and inspects clusters of coevolving residues by mapping them onto sequences, alignments or structures of choice, greatly simplifying downstream analysis steps. A rich and interactive graphic interface facilitates the biological interpretation of the results

Molecular basis of the interaction of the human tyrosine phosphatase PTPN3 with the hepatitis B virus core protein

International audienceInteractions between the hepatitis B virus core protein (HBc) and host cell proteins are poorly understood, although they may be essential for the propagation of the virus and its pathogenicity. HBc has a C-terminal PDZ (PSD-95, Dlg1, ZO-1)-binding motif (PBM) that is responsible for interactions with host PDZ domain-containing proteins. In this work, we focused on the human protein tyrosine phosphatase non-receptor type 3 (PTPN3) and its interaction with HBc. We solved the crystal structure of the PDZ domain of PTPN3 in complex with the PBM of HBc, revealing a network of interactions specific to class I PDZ domains despite the presence of a C-terminal cysteine in this atypical PBM. We further showed that PTPN3 binds the HBc protein within capsids or as a homodimer. We demonstrate that overexpression of PTPN3 significantly affects HBV infection in HepG2 NTCP cells. Finally, we performed proteomics studies on both sides by pull-down assays and screening of a human PDZ domain library. We identified a pool of human PBM-containing proteins that might interact with PTPN3 in cells and that could be in competition with the HBc PBM during infection, and we also identified potential cellular partners of HBc through PDZ-PBM interactions. This study opens up many avenues of future investigations into the pathophysiology of HBV