Thèse la Naissance du Sexe Retracer ses Origines Moléculaires Depuis nos Ancêtres Microbiens H/F - 75

Établissement : Université Paris-Saclay GS Life Sciences and Health
École doctorale : Structure et Dynamique des Systèmes Vivants
Laboratoire de recherche : Génomique métabolique - DRF/JACOB/Génoscope
Direction de la thèse : Violette DA CUNHA ORCID 0000000290357825
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-03-23T23:59:59

CECI EST UNE TRADUCTION AUTOMATIQUE DU TEXTE EN ANGLAIS, IL EST PREFERABLE DE LIRE LA VERSION ORIGINALE EN ANGLAIS
La reproduction sexuelle est une caractéristique clé de nombreux eucaryotes, mais pas des procaryotes, des bactéries ou des archées. En revanche, les bactéries et les archées peuvent échanger de l'ADN lors d'un processus appelé recombinaison. Étant donné que ces deux processus présentent des niveaux de complexité très différents et impliquent principalement des acteurs moléculaires distincts, nous ne comprenons pas encore pleinement comment ils sont liés et, par conséquent, comment la reproduction sexuelle a évolué.
La recombinaison homologue méiotique est l'étape clé de la reproduction sexuelle, conduisant à la production de gamètes haploïdes, et est donc essentielle à la fertilité de la plupart des eucaryotes [1]. Elle est initiée par le complexe TOPOVIL, qui est apparenté sur le plan évolutif à la topoisomérase de l'ADN archéenne, la topoisomérase VI (Topo VI) [2, 3, 4]. Cette dernière est présente chez toutes les archées, où elle joue un rôle essentiel dans la régulation de la topologie de l'ADN en relâchant les super-enroulements de l'ADN. On ne comprend pas encore comment cette activité a été réorientée, au cours de l'évolution, vers la recombinaison homologue chez les eucaryotes.
La découverte des archées Asgard en 2017 et les études qui ont suivi [5, 6] ont suscité un vif intérêt dans la communauté scientifique, car il s'est avéré que ces organismes sont les parents connus les plus proches des eucaryotes, avec un ancêtre asgardien à l'origine de tous les eucaryotes actuels. L'étude des archées Asgard et de leurs machineries moléculaires est donc particulièrement pertinente pour comprendre la transition d'une cellule procaryote petite et relativement simple vers une cellule eucaryote bien plus complexe et volumineuse.
Grâce à la phylogénomique, le laboratoire hôte a récemment découvert que les génomes des Asgard codent de manière unique des variantes de Topo VI qui sont les plus étroitement apparentées à la TOPOVIL eucaryote. Ces enzymes uniques représentent probablement un 'chaînon manquant' longtemps recherché entre les topoisomérases archéennes et la TOPOVIL eucaryote. Ainsi, la Topo VI des Asgard constitue un système modèle particulièrement pertinent pour étudier les origines évolutives de la reproduction sexuelle.
Dans ce projet de thèse interdisciplinaire et international, le ou la candidat(e) caractérisera, par des approches computationnelles, biochimiques et structurales, les protéines Topo VI des archées Asgard. En intégrant ces données, le ou la candidat(e) reliera les détails moléculaires aux perspectives évolutives, apportant ainsi un éclairage sur l'origine du sexe, l'un des événements clés de l'évolution des eucaryotes.

References:
1. Hunter, N. (2015) . Cold Spring Harb. Perspect. Biol. 7, a016618.
2. Keeney, S., Giroux, C. N. & Kleckner, N. (1997). Cell 88, 375-384.
3. Robert, T., Nore, A., Brun, C., Maffre, C., Crimi, B., Bourbon, H. M., & de Massy, B. (2016). Science, 351(6276), 943-949. https://doi.org/10.1126/science.aad5309
4. Bergerat, A., de Massy, B., Gadelle, D., Varoutas, P. C., Nicolas, A., & Forterre, P. (1997). Nature, 386(6623), 414-417. https://doi.org/10.1038/386414a0.
5. Zaremba-Niedzwiedzka, K., Caceres, E. F., Saw, J. H., Bäckström, D., Juzokaite, L., Vancaester, E., Seitz, K. W., Anantharaman, K., Starnawski, P., Kjeldsen, K. U., Stott, M. B., Nunoura, T., Banfield, J. F., Schramm, A., Baker, B. J., Spang, A., & Ettema, T. J. (2017). Nature, 541(7637), 353-358. https://doi.org/10.1038/nature21031
6. Liu, Y., Makarova, K. S., Huang, W. C., Wolf, Y. I., Nikolskaya, A. N., Zhang, X., Cai, M., Zhang, C. J., Xu, W., Luo, Z., Cheng, L., Koonin, E. V., & Li, M. (2021). Nature, 593(7860), 553-557. https://doi.org/10.1038/s41586-021-03494-3

The PhD candidate, Margot LAURENT, will work under the co-supervision of Dr. Violette da CUNHA (CPJ, Université d'Evry Paris-Saclay) and Dr. Tamara BASTA (Ass. Prof, Université Paris-Saclay). V. da CUNHA and T. BASTA have successfully co-supervised two PhD students in the past (Pichard-Kostuch et al., 2023; Villain et al., 2022, see reference list below). The PhD candidate will also collaborate with Dr. Bertram DAUM (Exeter University, UK) for the structural analysis of Asgard Topo VI.

Violette da CUNHA is a confirmed expert in phylogenomics with special interest in genome evolution. She has analyzed the phylogeny of numerous archaeal proteins including DNA topoisomerases (see reference list below). She recently supervised a M1 intern who worked on phylogeny of Asgard Topo VI. She has obtained Junior Professorship Chair in 2023 and started her own group within LABGeM (Genoscope). The LABGeM research activity is certified as FAIR (Findable, Accessible, Interoperable, Reusable) science.
Tamara BASTA is head of the DNA topology in Archaea team at the I2BC institute. She is internationally recognized for her biochemical studies of fundamental molecular mechanisms in archaea, including DNA topoisomerases (see reference list below). She has supervised numerous interns, three PhD students, and is currently supervisor of two first-year PhD candidates (one main supervisor and one co-supervisor). In addition to T. BASTA, Margot's work will be supported by Dr. Sylvie AUXILIEN, research engineer in T. BASTA's team.

Bertram DAUM is head of an internationally leading team in the structural biology of archaea (see reference list below). He is already collaborating with T. BASTA's team through the Paris-Saclay - Exeter Accelerator grant. B. DAUM has direct access to Talos Arctica Transmission Electron Microscope (TEM) and Titan Krios TEMs through the lab's block allocated access at the National CryoEM Facility at eBIC (Harwell, Oxfordshire). This equipment will be used to record high resolution cryoEM data and process them through established single particle processing pipelines.

References:

Da CUNHA
Pichard-Kostuch A, Da Cunha V, Oberto J, Sauguet L and Basta T (2023) The universal Sua5/TsaC family evolved different mechanisms for the synthesis of a key tRNA modification. Front. Microbiol. 14:1204045.doi: 10.3389/fmicb.2023.1204045
Da Cunha V*#, Gaïa M*, Ogata H. Jaillon O., Delmont T.O., Forterre P. Giant Viruses Encode Actin-Related Proteins. Mol Biol Evol. 2022 Feb 3;39(2):msac022.
Villain, P., Catchpole, R., Forterre, P., Oberto, J., da Cunha, V., & Basta, T. (2022). Expanded Dataset Reveals the Emergence and Evolution of DNA Gyrase in Archaea. Molecular biology and evolution, 39(8), msac155. https://doi.org/10.1093/molbev/msac155
Badel C*, Da Cunha V*, Oberto J. Archaeal tyrosine recombinases. FEMS Microbiol Rev. 2021 Feb 1.
Takahashi T., Da Cunha V., Krupovic M., Mayer C., Forterre P., Gadelle D. Expanding the type IIB DNA topoisomerase family: identification of new topoisomerase and topoisomerase-like proteins in mobile genetic elements. NAR Genomics and Bioinformatics 2020 March. 2(1).

BASTA
Perrochia L, Crozat E, Hecker A, Zhang W, Bareille J, Collinet B, van Tilbeurgh H, Forterre P, Basta T. (2013). In vitro biosynthesis of a universal t6A tRNA modification in Archaea and Eukarya. Nucleic Acids Res. 41(3):1953-64. doi: 10.1093/nar/gks1287.
Perrochia L, Guetta D, Hecker A, Forterre P, Basta T. Functional assignment of KEOPS/EKC complex subunits in the biosynthesis of the universal t6A tRNA modification. (2013). Nucleic Acids Res. 41(20):9484-99. doi: 10.1093/nar/gkt720.
Daugeron MC, Missoury S, Da Cunha V, Lazar N, Collinet B, van Tilbeurgh H, Basta T. (2023). A paralog of Pcc1 is the fifth core subunit of the KEOPS tRNA-modifying complex in Archaea. Nat Commun. 14(1):526. doi: 10.1038/s41467-023-36210-y.
Villain, P., da Cunha, V., Villain, E., Forterre, P., Oberto, J., Catchpole, R., & Basta, T. (2021). The hyperthermophilic archaeon Thermococcus kodakarensis is resistant to pervasive negative supercoiling activity of DNA gyrase. Nucleic acids research, 49(21), 12332-12347. https://doi.org/10.1093/nar/gkab869
Villain, P., Catchpole, R., Forterre, P., Oberto, J., da Cunha, V., & Basta, T. (2022). Expanded Dataset Reveals the Emergence and Evolution of DNA Gyrase in Archaea. Molecular biology and evolution, 39(8), msac155. https://doi.org/10.1093/molbev/msac155

DAUM
Daum B, Vonck J, Bellack A, Chaudhury P, Reichelt R, Albers SV, Rachel R, Kühlbrandt W. Structure and in situ organisation of the Pyrococcus furiosus archaellum machinery. Elife. 2017 Jun 27;6:e27470. doi: 10.7554/eLife.27470. PMID: 28653905; PMCID: PMC5517150.
Gambelli L, Meyer BH, McLaren M, Sanders K, Quax TEF, Gold VAM, Albers SV, Daum B. Architecture and modular assembly of Sulfolobus S-layers revealed by electron cryotomography. Proc Natl Acad Sci U S A. 2019 Dec 10;116(50):25278-25286. doi: 10.1073/pnas.1911262116. Epub 2019 Nov 25. PMID: 31767763; PMCID: PMC6911244.
Gambelli L, Isupov MN, Conners R, McLaren M, Bellack A, Gold V, Rachel R, Daum B. An archaellum filament composed of two alternating subunits. Nat Commun. 2022 Feb 7;13(1):710. doi: 10.1038/s41467-022-28337-1. PMID: 35132062; PMCID: PMC8821640.
Gaines MC, Isupov MN, Sivabalasarma S, Haque RU, McLaren M, Mollat CL, Tripp P, Neuhaus A, Gold VAM, Albers SV, Daum B. Electron cryo-microscopy reveals the structure of the archaeal thread filament. Nat Commun. 2022 Dec 1;13(1):7411. doi: 10.1038/s41467-022-34652-4. PMID: 36456543; PMCID: PMC9715654.
McLaren M, Conners R, Isupov MN, Gil-Díez P, Gambelli L, Gold VAM, Walter A, Connell SR, Williams B, Daum B. CryoEM reveals that ribosomes in microsporidian spores are locked in a dimeric hibernating state. Nat Microbiol. 2023 Oct;8(10):1834-1845. doi: 10.1038/s41564-023-01469-w. Epub 2023 Sep 14. PMID: 37709902; PMCID: PMC10522483.
Gambelli L, McLaren M, Conners R, Sanders K, Gaines MC, Clark L, Gold VAM, Kattnig D, Sikora M, Hanus C, Isupov MN, Daum B. Structure of the two-component S-layer of the archaeon Sulfolobus acidocaldarius. Elife. 2024 Jan 22;13:e84617. doi: 10.7554/eLife.84617. PMID: 38251732; PMCID: PMC10903991.
Gaines MC, Sivabalasarma S, Isupov MN, Haque RU, McLaren M, Hanus C, Gold VAM, Albers SV, Daum B. CryoEM reveals the structure of an archaeal pilus involved in twitching motility. Nat Commun. 2024 Jun 14;15(1):5050. doi: 10.1038/s41467-024-45831-w. PMID: 38877033; PMCID: PMC11178815.
Gaines MC, Isupov MN, McLaren M, Mollat CL, Haque RU, Stephenson JK, Sivabalasarma S, Hanus C, Kattnig D, Gold VAM, Albers S, Daum B. Towards a molecular picture of the archaeal cell surface. Nat Commun. 2024 Nov 29;15(1):10401. doi: 10.1038/s41467-024-53986-9. PMID: 39614099; PMCID: PMC11607397.
Gaines MC, Isupov MN, McLaren M, Haque RU, Recalde A, Bargiela R, Gold VAM, Albers SV, Golyshin PN, Golyshina OV, Daum B. Unusual cell surfaces, pili, and archaella of Thermoplasmatales archaea. ISME J. 2025 Jan 2;19(1):wraf176. doi: 10.1093/ismejo/wraf176. PMID: 40801261; PMCID: PMC12448462.
Zhang DX, Isupov MN, Davies RM, Schwarzer S, McLaren M, Stuart WS, Gold VAM, Oksanen HM, Quax TEF, Daum B. Cryo-EM resolves the structure of the archaeal dsDNA virus HFTV1 from head to tail. Sci Adv. 2025 Oct 3;11(40):eadx1178. doi: 10.1126/sciadv.adx1178. Epub 2025 Oct 3. PMID: 41042861; PMCID: PMC12494034.

The first goal will be to build a complete picture of the diversity of Asgard Topo VI and eukaryotic TOPOVIL complexes, elucidating the evolutionary relationships between archaeal and eukaryotic variants. The sequence similarity shared across these proteins is low, thus necessitating the use of structural phylogenetics methods. This will allow identification of signatures that distinguish eukaryotic and archaeal TOPOVIL, and, by integrating the data from the biochemical and structural analysis, relating these to their function and evolution.
The second goal will be to express and purify functional Asgard Topo VI, selected based on sequence and structural similarity to eukaryotic TOPOVIL. DNA cleavage and unwinding activity of these recombinant Asgard Topo VI will be reconstituted and the products characterized on agarose gels and by sequencing. This will reveal whether Asgard Topo VI can induce recombination and how the sequence, bendability and topology of the DNA substrate affects activity.
The third goal will be to determine the structure of the Asgard Topo VI with and without DNA using cryoEM. This will provide atomic-level insights into the Asgard Topo VI DNA processing mechanism. The structural data will be compared with existing structures of eukaryotic homologs to infer their molecular evolution.

In V. da CUNHA's team the PhD candidate will use PanGBank database (maintained by the LABGeM team) gathering pangenomes of all prokaryotes. The database will be updated at the beginning of the project with a non-redundant set of Asgard archaeal genomes. The pangenomic approach will allow (i) to considerably reduce the bias of using a single representative genome per species or genus and (ii) to eliminate redundancy by removing strain-specific genes. The candidate will thencreate profile hidden Markov models (HMM) of typical archaeal Topo VI and match them against the Asgards pangenomes present in the PanGBank database to generate presence-absence' phyletic patterns across the Asgard diversity. Asgard Topo VI predicted from the homology searches, and HMM profile search, will be used to perform protein similarity network and phylogenetic analysis and, in fine, to classify these enzymes into several subgroups. Structural models for subgroup-representative Asgard Topo VI will be predicted using AlphaFold3 and ESMFold. Finally, the candidate will infer the evolutionary history of Asgard Topo VI within the archaeal, eukaryotic, bacterial and viral ones using phylogenetic tree construction. Another outcome of this work will be to select a panel of Asgard Topo VI proteins, based on their sequence and structural similarity to eukaryotic TOPOVIL.

In T. BASTA's team, the candidate will express and purify the selected panel of Asgard Topo VI proteins.
The genes encoding Topo VI will be codon-optimized (GenScript) and cloned into pET28a plasmid such that the protein can be expressed with a TEV protease-cleavable His6 tag. The cassette will be expressed in E. coli and purified using a three-step liquid chromatography (IMAC column, heparin column and gel filtration column) using protocols already established in T. BASTA's team. In addition to gel filtration the candidate will use SEC-MALS (I2BC's Interactions of Macromolecules facility) to precisely determine the molecular mass of the complexes in solution and deduce from there the subunit stoichiometry.
The activity of purified Topo VI and TOPOVIL-like complexes will be tested using standard agarose gel-based plasmid DNA relaxation, cleavage and decatenation assays already running in the T. BASTA's lab. In addition, we will measure the ATPase activity of purified complexes using a standard PK/LDH linked spectrophotometric assay. DNA binding will be evaluated using EMSA and Bio-Layer Interferometry (BLI) (I2BC's Interactions of Macromolecules facility). Altogether, these experiments will allow to (i) optimize the expression and purification of Asgard Topo VI; and (ii) determine basic enzymatic properties of Asgard Topo VI such that they can be compared to classical archaeal Topo VI and eukaryotic TOPOVIL.

The purified Asgard Topo VI will be sent to B. DAUM's lab where sample preparation for cryo-EM and imaging conditions will be optimized using previously established protocols. It is also planned that the candidate will spend several weeks in B. DAUM's lab such that she can learn how to prepare the samples, collect the data and build atomic models of the Topo VI proteins via cutting-edge AI-guided approaches.