Roxane Lestini
Professor - Ecole polytechnique
Pièce : 84-20 12
Tél :+33 (0) 16933 5028
roxane.lestini @polytechnique.edu
ORCID ID: 0000-0001-7377-4734
Google Scholar Account
Scientific Interests
We aim at unravelling the molecular features of genome maintenance and replication restart in archaea. Replication restart of arrested replication forks is crucial for genome maintenance and cell viability. It is the focus of studies from bacteria to human to understand the different pathways and molecular mechanisms involved. Studies in bacteria revealed a close link between replication and homologous recombination. Indeed, arrested replication forks can be processed by recombination proteins, and replication can restart from recombination intermediates. That link between replication and homologous recombination is also conserved in yeast and higher eukaryotes. Moreover, a link between replicative stress and associated genomic instability and cancer predisposition has been established in a number of human disorders such as Bloom’s syndrome and Fanconi’s anemia. However, little is known concerning this important process in archaea.
Archaeal main chromosome is circular, a bacterial-like feature, but often contains multiple active replication origins, a eukaryotic-like feature. This is notably the case of the archaea Haloferax volcanii. However, despite the presence of four replication origins on its main chromosome, we have shown by genetic studies that replication restart pathways are essential for viability. Thus, H. volcanii is a relevant model organism to investigate replication restart pathways in archaeal cells. Haloferax volcanii is a halophilic archaea easily grown in laboratory (120 minutes generation time at 45°C on rich media) for which powerful tools have been developed. This includes genetic tools and the use of protein labeling by the green fluorescent protein GFP to study their localization in living cells, which are essential to tackle the in vivo study of fundamental processes such as DNA replication.
We have been interested in understanding the role of the archaeal Hef protein, a member of the XPF/MUS81 family of endonucleases. This family is also found in eukaryotes and play a critical role in DNA repair and replication fork restart. Among this large and intensively studied nuclease family, archaeal Hef proteins are unique as they contain active helicase and nuclease domains that in vitro are required for the rearrangement of fork structured DNA. By genetic studies, we have shown that both the helicase and nuclease activities of Hef are essential for cell viability in the absence of proteins involved in homologous recombination: the Holliday junction resolvase Hjc, an endonuclease acting on four-branched DNA structures, or the RadA recombinase. We have proposed that Hef and Hjc/RadA provide alternative means to restart stalled DNA replication forks in archaea (figure 1) (Lestini et al., 2010).
To further understand the physiological role of Hef in genome stability, we have studied its localization by fluorescence microscopy live-cell imaging coupled to quantitative image analysis. For that purpose, we have expressed a functional Hef protein fused to the Green Fluorescent Protein (GFP) from its native chromosomal locus. We have demonstrated that Hef proteins are dynamically recruited to arrested replication forks in response to DNA replication stress and proposed Hef may enhance replication fork stability by directly interacting with collapsed replication forks (figure 2) (Lestini et al., 2013).
We now aim at identifying proteins involved in replication restart by characterizing the interaction network of Hef in H. volcanii. These experiments will provide key understanding of replisome apparatus at active replication forks and proteins specifically recruited at arrested/stalled replication forks to allow replication restart.
PhD and PhD Proposals
- PhD of Dorian Noury (2022-2025) to study replication dynamics in polyploid archaeal cells at the single molecule level - Co-direction with Nicolas Olivier, LOB
- PhD proposal: Unraveling replication restart in the archaea Haloferax volcanii (Full proposal)
Background
2022-present: Professor at Laboratory for Optics and Biosciences, CNRS / INSERM / Ecole Polytechnique (Palaiseau, France)
2014-2022: Assistant professor at Laboratory for Optics and Biosciences, CNRS / INSERM / Ecole Polytechnique (Palaiseau, France)
2012-2014: Chargé d'enseignement at Laboratory for Optics and Biosciences, CNRS / INSERM / Ecole Polytechnique (Palaiseau, France)
2009-2012: Post-doctoral position at Laboratory for Optics and Biosciences, CNRS / INSERM / Ecole Polytechnique (Palaiseau, France)
2008-2009: Post-doctoral position at Nottingham University (UK) in Dr. Thosten Allers laboratory
2004-2008: PhD on replication fork restart in E. coli at the CGM, CNRS (Gif-sur-yvette, France), supervised by Dr. Bénédicte Michel
Teaching
TD (Tutorial class) "Biologie moléculaire et information génétique" (BIO452) for 2nd year student at Ecole polyetchnique
MODAL (Practical Course) for 2nd year student at Ecole polyetchnique untitled « Bioluminescence et GFP »
TREX (Practical Course) for 3rd year student at Ecole polyetchnique untitled « Rôle des flagellines dans la motilité chez l’archée Haloferax volcanii »
Master 2 Biologie Santé, Parcours Microbiologie Fondamentale – Paris XI/Paris Saclay « Une révolution récente en (micro)biologie : les archées »
Licence 3 Sciences de la vie - Université Versailles-Saint Quentin « Réplication et Conversion génique chez les archées »
Publications (ORCID ID: 0000-0001-7377-4734)
Lestini R., Collien Y., Olivier D., Olivier N., Myllykallio, H. (2022). BrdU Incorporation and Labeling of Nascent DNA to Investigate Archaeal Replication Using Super-Resolution Imaging. Methods Mol Biol. 2522:419-434. DOI
Cockram C., Thierry A., Gorlas A., Lestini R., Koszul R. (2021). Euryarchaeal genomes are folded into SMC-dependent loops and domains, but lack transcription-mediated compartmentalization. Mol Cell. 81(3):459-472.e10. DOI
Sodolescu A., Dian C., Terradot L., Bouzhir-Sima L., Lestini R., Myllykallio H., Skouloubris S., Liebl U. (2018). Structural and functional insight into serine hydroxymethyltransferase from Helicobacter pylori. PloS one, 13(12), e0208850. DOI
Delpech F., Collien Y., Mahou P., Beaurepaire E., Myllykallio M., Lestini R. Snapshots of archaeal DNA replication and repair in living cells using super-resolution imaging, Nucleic Acids Research, DOI
van Tran N., Muller L., Ross R. L., Lestini R., Létoquart J., Ulryck N., Limbach P. A., de Crécy-Lagard V., Cianférani S., Graille M. (2018). Evolutionary insights into Trm112-methyltransferase holoenzymes involved in translation between archaea and eukaryotes. Nucleic acids research, 46(16), 8483-8499, DOI
Sakina K., Lestini R., Myllykallio H. and Houali K. Isolation and identifcation of two extremely halophilic archaea from sebkhas in the Algerian Sahara (2018) Cellular and molecular biology 64(4):83-91
Plateau, P., Saveanu, C., Lestini, R., Dauplais, M., Decourty, L., Jacquier, A., Blanquet, S. and Lazard, M. (2017) Exposure to selenomethionine causes selenocysteine misincorporation and protein aggregation in Saccharomyces cerevisiae. Sci Rep, 7, 44761, DOI
Becker, H.F., Heliou, A., Djaout, K., Lestini, R., Regnier, M. and Myllykallio, H. (2017) High-throughput sequencing reveals circular substrates for an archaeal RNA ligase. RNA Biol, 1-11. DOI
Lestini, R., Delpech, F. and Myllykallio, H. (2015) DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii. Biochimie, 118, 254-263. DOI
Lestini, R., Delpech, F. and Myllykallio, H. (2015) DNA Replication Restart in Archaea, Advances in DNA Repair, Prof. Clark Chen (Ed.), InTech, DOI
Rezgui, R., Lestini, R., Kuhn, J., Fave, X., McLeod, L., Myllykallio, H., Alexandrou, A. and Bouzigues, C. (2014) Differential interaction kinetics of a bipolar structure-specific endonuclease with DNA flaps revealed by single-molecule imaging. PLoS One, 9, e113493. DOI
Lestini, R., Laptenok, S.P., Kuhn, J., Hink, M.A., Schanne-Klein, M.C., Liebl, U. and Myllykallio, H. (2013) Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication. Nucleic Acids Res, 41, 10358-10370. DOI
Creze, C., Ligabue, A., Laurent, S., Lestini, R., Laptenok, S.P., Khun, J., Vos, M.H., Czjzek, M., Myllykallio, H. and Flament, D. (2012) Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels. J Biol Chem, 287, 15648-15660. DOI
Lestini, R., Creze, C., Flament, D., Myllykallio, H., and Henneke, G., (2011). Archaeal DNA Repair Nucleases, DNA Repair - On the Pathways to Fixing DNA Damage and Errors, Francesca Storici (Ed.), InTech, DOI
Creze, C., Lestini, R., Kuhn, J., Ligabue, A., Becker, H.F., Czjzek, M., Flament, D. and Myllykallio, H. (2011) Structure and function of a novel endonuclease acting on branched DNA substrates. Biochem Soc Trans, 39, 145-149. DOI
Lestini, R., Duan, Z. and Allers, T. (2010) The archaeal Xpf/Mus81/FANCM homolog Hef and the Holliday junction resolvase Hjc define alternative pathways that are essential for cell viability in Haloferax volcanii. DNA Repair (Amst), 9, 994-1002. DOI
Baharoglu, Z.*, Lestini, R.*, Duigou, S. and Michel, B. (2010) RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases. Mol Microbiol, 77, 324-336 *co-premier auteur DOI
Lestini, R. and Michel, B. (2008) UvrD and UvrD252 counteract RecQ, RecJ, and RecFOR in a rep mutant of Escherichia coli. J Bacteriol, 190, 5995-6001. DOI
Centore, R.C., Lestini, R. and Sandler, S.J. (2008) XthA (Exonuclease III) regulates loading of RecA onto DNA substrates in log phase Escherichia coli cells. Mol Microbiol, 67, 88-101. DOI
Michel, B., Boubakri, H., Baharoglu, Z., LeMasson, M. and Lestini, R. (2007) Recombination proteins and rescue of arrested replication forks. DNA Repair (Amst), 6, 967-980. DOI
Michel, B., Baharoglu, Z., and Lestini, R. (2007) Genetics of recombination in the model bacteriun Escherichia coli. Topics in Current Genetics, Aguilera A. and Rothstein R. eds, Springer.
Lestini, R. and Michel, B. (2007) UvrD controls the access of recombination proteins to blocked replication forks. EMBO J, 26, 3804-3814. DOI
Janniere, L., Canceill, D., Suski, C., Kanga, S., Dalmais, B., Lestini, R., Monnier, A.F., Chapuis, J., Bolotin, A., Titok, M. et al. (2007) Genetic evidence for a link between glycolysis and DNA replication. PLoS One, 2, e447. DOI
Bidnenko V., Lestini R., Michel B.The Escherichia coli UvrD helicase is essential for Tus removal during recombination-dependent replication restart from Ter sites.Mol Microbiol. 2006 Oct;62(2):382-96.DOI