Découverte et études structure-fonction de facteurs-clés nécessaires à ls synthèse d'ADN non-standard ZTGC observé dans la famille Siphoviridae

The subject for this thesis is to dissect the enzymatic pathway allowing a non-canonical base 2-aminoadenine, or diaminopurine (Z) to replace adenine (A) in the genomes of a number of Siphoviridae bacteriophages. 2-aminoadenine and thymine (T) form the Z:T pair bound by fully saturated triple hydrogen bond. Together with the standard G:C pair they form ZTGC-DNA which is resistant to host’s restriction enzymes. I first focus on cyanophage S-2L, the originally-described bearer of 2-aminoadenine. I identify a DNA primase-polymerase, PrimPol, responsible for its replication, with surprisingly similar activity towards dATP and dZTP. This prompted the characterization of a dATP-specific triphosphatase, DatZ. Its activity and conservation between the phages of interest explains the mechanism behind adenine removal. Secondly, I find that PurZ of phage S-2L’s, a key enzyme in diaminopurine production, is not only an ATPase but also a dATPase. I identify a nucleotide pyrophosphatase, MazZ, as an essential component of the conserved Z biosynthetic pathway, that converts dGTP into dGMP, thus generating one of the substrates of PurZ. High resolution crystallographic structures of all 4 enzymes with their respective ligands explain the specificities observed in catalytic tests - or lack thereof. Finally, I characterized the structure of a Z-specific family A DNA polymerase, PolZ, found in a related vibriophage φVC8 but absent in S-2L. Its crystallographic structure in polymerase-exonuclease “coupled-open” and “coupled-close” states offers an explanation for the observed specificity.Le but de cette thèse est de décrire le chemin métabolique permettant de remplacer l’adénine (A) par la 2-aminoadénine (Z) dans le génome de bactériophages Siphoviridae. La 2-aminoadénine et la thymine (T) forment la paire Z:T, liée par trois liaisons hydrogène. Avec la paire G:C classique, ils forment un ADN « ZTGC » qui est résistant aux enzymes de réstriction de l’hôte. En premier lieu, mes travaux se sont focalisés sur le cyanophage S-2L, décrit comme porteur de 2-aminoadénine. J’ai d’abord étudié la primase-polymérase, PrimPol, responsable de la réplication de l’ADN chez ces phages, et qui possède la capacité surprenante d’incorporer à la fois le dATP et le dZTP. J’ai ensuite caractérisé une triphosphatase dATP-spécifique, appelée DatZ, conservée chez tous les bactériophages Siphoviridae, responsable de la dégradation spécifique du dATP. J’ai également mis en évidence que PurZ, l’enzyme-clé pour la production de la diaminopurine est non seulement une ATPase mais aussi une dATPase. J’ai identifié une nucléotide pyrophosphatase, appelée MazZ, composant essentiel du chemin de biosynthèse de Z, qui convertit dGTP en dGMP, en générant ainsi un des substrats de PurZ. Structures cristallographiques de haute résolution de tous les 4 enzymes avec leurs ligands respectifs expliquent les spécificités observées dans les tests catalytiques – ou leur absence. Enfin, j’ai caractérisé une structure d’une ADN polymérase Z-spécifique de famille A, PolZ, trouvée dans le vibriophage φVC8, mais absente dans S-2L. J’ai résolu sa structure cristallographique en modes polymérase et exonuléase « couplé-ouvert » et « couplé-fermé », permettant de decrire son activité au niveau atomique

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

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

International audienceAll genetic information in cellular life is stored in DNA copolymers composed of four basic building blocks (ATGC-DNA). In contrast, a group of bacteriophages belonging to families Siphoviridae and Podoviridae has abandoned the usage of one of them, adenine (A), replacing it with 2-aminoadenine (Z). The resulting ZTGC-DNA is more stable than its ATGC-DNA counterpart, owing to the additional hydrogen bond present in the 2-aminoadenine:thymine (Z:T) base pair, while the additional amino group also confers resistance to the host endonucleases. Recently, two classes of replicative proteins found in ZTGC-DNAcontaining phages were characterized and one of them, DpoZ from DNA polymerase A (PolA) family, was shown to possess significant Z-vs-A specificity. Here, we present the crystallographic structure of the apo form of DpoZ of vibriophage VC8, composed of the 3-5 exonuclease and polymerase domains. We captured the enzyme in two conformations that involve the tip of the thumb subdomain and the exonuclease domain. We highlight insertions and mutations characteristic of VC8 DpoZ and its close homologues. Through mutagenesis and functional assays we suggest that the preference of VC8 DpoZ towards Z relies on a polymerase backtracking process, more efficient when the nascent base pair is A:T than when it is Z:T

Characterization of a triad of genes in cyanophage S-2L sufficient to replace adenine by 2-aminoadenine in bacterial DNA

International audienceCyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthetase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene ( mazZ) located between purZ and datZ genes in S-2L and related phage genomes. We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes – datZ , mazZ and purZ – was expressed in E. coli , resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA

How cyanophage S-2L rejects adenine and incorporates 2-aminoadenine to saturate hydrogen bonding in its DNA

International audienceBacteriophages have long been known to use modified bases in their DNA to prevent cleavage by the host's restriction endonucleases. Among them, cyanophage S-2L is unique because its genome has all its adenines (A) systematically replaced by 2-aminoadenines (Z). Here, we identify a member of the PrimPol family as the sole possible polymerase of S-2L and we find it can incorporate both A and Z in front of a T. Its crystal structure at 1.5 Å resolution confirms that there is no structural element in the active site that could lead to the rejection of A in front of T. To resolve this contradiction, we show that a nearby gene is a triphosphohydolase specific of dATP (DatZ), that leaves intact all other dNTPs, including dZTP. This explains the absence of A in S-2L genome. Crystal structures of DatZ with various ligands, including one at sub-angstrom resolution, allow to describe its mechanism as a typical two-metal-ion mechanism and to set the stage for its engineering

Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli

International audienceTo stop the COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which caused more than 2.5 million deaths to date, new antiviral molecules are urgently needed. The replication of SARS-CoV-2 requires the RNA-dependent RNA polymerase (RdRp), making RdRp an excellent target for antiviral agents. RdRp is a multi-subunit complex composed of 3 viral proteins named nsp7, nsp8 and nsp12 that ensure the ~30 kb RNA genome's transcription and replication. The main strategies employed so far for the overproduction of RdRp consist of expressing and purifying the three subunits separately before assembling the complex in vitro. However, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells. Here, we describe an alternative strategy to co-express the full SARS-CoV-2 RdRp in E. coli, using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp shows that it forms a complex with the expected (nsp7)(nsp8)2(nsp12) stoichiometry. RNA polymerization activity was measured using primer-extension assays showing that the purified enzyme is functional. The purification protocol can be achieved in one single day, surpassing in speed all other published protocols. Our construct is ideally suited for screening RdRp and its variants against very large chemical compounds libraries and has been made available to the scientific community through the Addgene plasmid depository (Addgene ID: 165451)

Meet-U: Educating through research immersion.

We present a new educational initiative called Meet-U that aims to train students for collaborative work in computational biology and to bridge the gap between education and research. Meet-U mimics the setup of collaborative research projects and takes advantage of the most popular tools for collaborative work and of cloud computing. Students are grouped in teams of 4-5 people and have to realize a project from A to Z that answers a challenging question in biology. Meet-U promotes "coopetition," as the students collaborate within and across the teams and are also in competition with each other to develop the best final product. Meet-U fosters interactions between different actors of education and research through the organization of a meeting day, open to everyone, where the students present their work to a jury of researchers and jury members give research seminars. This very unique combination of education and research is strongly motivating for the students and provides a formidable opportunity for a scientific community to unite and increase its visibility. We report on our experience with Meet-U in two French universities with master's students in bioinformatics and modeling, with protein-protein docking as the subject of the course. Meet-U is easy to implement and can be straightforwardly transferred to other fields and/or universities. All the information and data are available at www.meet-u.org

Examples of strategies and results for the 2016–2017 edition.

<p>Left panel: Team B implemented an efficient sampling algorithm using a grid representation of the proteins to be docked and FFT. For the scoring, they used evolutionary information extracted from multiple sequence alignments of homologs of the two partners. Right panel: Team D used biological knowledge during the sampling step to filter out conformations early and drastically reduce the search space. The results obtained by the students (Teams B and D) on two complexes (barnase–barstar complex, Protein Data Bank [PDB] code: 1AY7, and an antibody–antigen complex, PDB code: 1JPS, respectively) are comparable to those obtained from state-of-the-art methods, namely ZDOCK [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005992#pcbi.1005992.ref010" target="_blank">10</a>] and ATTRACT [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005992#pcbi.1005992.ref011" target="_blank">11</a>]. ZDOCK relies on efficient sampling using FFT and on an optimized energy function [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005992#pcbi.1005992.ref010" target="_blank">10</a>]. ATTRACT proceeds through minimization steps using an empirical, coarse-grained molecular mechanics potential [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005992#pcbi.1005992.ref011" target="_blank">11</a>]. Candidate conformations for the complexes are represented as cartoons and superimposed onto the known crystallographic structures. The receptor is in black, the ligand from the candidate conformation is colored (in orange for Meet-U students, blue for ZDOCK, and purple for ATTRACT), and that from the crystallographic structure is in grey. With each candidate conformation are associated its rank, according to the scoring function of the method, and its deviation (in Å) from the crystallographic structure. FFT, Fast Fourier Transform; PDB, protein data bank.</p