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DNA repair in prokaryotes

DNA repair in prokaryotes

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DNA repair systems help maintain the integrity of genomes and therefore play a key role at both the cellular level and in the transmission of a faithful genetic inheritance to offspring. 

We are particularly interested in a protein called NucS (also known as EndoMS in the literature). The presence of this protein, discovered in the laboratory, is limited to the prokaryotic world. It is found in some species of Archaea (Crenarcheota and Euryarcheota) and in many bacteria belonging to the large phylum Actinobacteria (e.g. Corynebacteria and Mycobacteria). It exhibits cleavage activity against a variety of DNA substrates and is primarily involved in the detection and repair of mismatches.

Work is currently being undertaken within our team to better understand the mechanism by which NucS is involved in this repair. Two bacterial models are being studied in the lab: Corynebacterium glutamicum and Mycobacterium smegmatis. Various interdisciplinary approaches including molecular biology, genetics, biochemistry, microscopy and bioinformatics are used.

It has already been shown that NucS interacts with the protein known as beta-clamp (i.e. PCNA in archaea and DnaN in bacteria). Part of the project is therefore aimed at identifying and characterizing other partner proteins involved in this repair pathway. A second part of our work is focused on assessing the impact of mutations in the nucS gene on antibiotic resistance, which has been detected in numerous clinical isolates of the tuberculosis etiologic agent, Mycobacterium tuberculosis.

Figure. A) Dimeric 3D structure of the NucS protein from Thermococcus kodakarensis (PDB: 5GKE; 5GKJ). The protein in the absence of DNA has an "open" conformation (left) that closes in the presence of a mismatched substrate (right) (adapted from Nakae et al. (2016). Structure, 24:1960-71). B) In vitro cleavage activity of a DNA substrate with a G:T mismatch by the NucS protein. This activity is enhanced in the presence of the DnaN protein (beta-clamp). C) Colonies of C. glutamicum bacteria that have become resistant to rifampicin in a wild-type and DnucS mutant context. The spontaneous mutation rate is increased by a factor of 200 in the absence of NucS (adapted from Ishino et al. (2018) Nucleic acids research, 46: 6206-17).

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Keywords: microbiology, DNA repair, mismatches, NucS, beta-clamp, C. glutamicum, M. smegmatis, M. tuberculosis, antibiotic resistance.

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Publications

  • Ishino S, Skouloubris S, Kudo H, l'Hermitte-Stead C, Es-Sadik A, Lambry JC, Ishino Y, Myllykallio H. Nucleic Acids Res. 2018 Jul 6;46(12):6206-6217. https://doi.org/10.1093/nar/gky460

  • Rezgui R, Lestini R, Kühn J, Fave X, McLeod L, Myllykallio H, Alexandrou A, Bouzigues C. PLoS One. 2014. Nov 20;9(11):e113493. https://doi.org/10.1371/journal.pone.0113493

  • Creze C, Ligabue A, Laurent S, Lestini R, Laptenok SP, Khun J, Vos MH, Czjzek M, Myllykallio H, Flament D. J Biol Chem. 2012 May 4;287(19):15648-60. https://doi.org/10.1074/jbc.M112.346361

  • Creze C, Lestini R, Kühn J, Ligabue A, Becker HF, Czjzek M, Flament D, Myllykallio H. Structure and function of a novel endonuclease acting on branched DNA substrates. Biochem Soc Trans. 2011 Jan;39(1):145-9. https://doi.org/10.1042/BST0390145

  • Ren B, Kühn J, Meslet-Cladiere L, Briffotaux J, Norais C, Lavigne R, Flament D, Ladenstein R, Myllykallio H. Structure and function of a novel endonuclease acting on branched DNA substrates. EMBO J. 2009 Aug 19;28(16):2479-89. https://doi.org/10.1038/emboj.2009.192

  • Ren B, Kuhn J, Meslet-Cladiere L, Myllykallio H, Ladenstein R. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007 May 1;63(Pt 5):406-8. https://doi.org/10.1107/S1744309107015278

  • Meslet-Cladiére L, Norais C, Kuhn J, Briffotaux J, Sloostra JW, Ferrari E, Hübscher U, Flament D, Myllykallio H. J Mol Biol. 2007. Oct 5;372(5):1137-48. https://doi.org/10.1016/j.jmb.2007.06.056 

Collaborations

  • Dr Guislaine Refrégier, IDEEV, UniV. Paris-Saclay
  • Pr Emmanuelle Cambau, University Paris Cite - APHP (Bichat Claude-Bernard hospital)
  • Pr Lars Jelsbak, DTU, Danemark
  • Pr Pierre Leblond, Univ. de Lorraine, Nancy
  • Didier Flament, IFREMER, Brest

Fundings

  • ANR-22-CE12-0042 / Avril 2023, durée 48 mois
  • ANRS – Maladies infectieuses émergentes Agence autonome de l’INSERM (EMERGENCES N°23534)