Vers la compréhension des mécanismes de réparation de l'ADN chez Streptomyces : identification d'acteurs de la recombinaison

Abstract : Double strand breaks (DSB) constitute the most deleterious form of DNA damage that a bacterial cell can encounter. Two major pathways can carry out DSB repair in bacteria: homologous recombination and Non-Homologous End Joining (NHEJ). Streptomyces is a model bacterium to explore the relative impact of these recombination mechanisms on genome structure and evolution; the chromosome is indeed typified by its linearity, its compartmentalized genetic organization and its remarkable genomic plasticity. The objective of this research is to identify actors involved in DSB repair mechanisms which remain mostly elusive in Streptomyces up to now. The first step of DSB repair by homologous recombination is the resection of broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB and Mycobacterium tuberculosis AdnAB. In silico analysis of Streptomyces genomes allowed to identify homologues for adnA and adnB which constitute a highly conserved locus within the genus. Attempts to disrupt these two genes were unsuccessful in Streptomyces ambofaciens as well as in Streptomyces coelicolor, unless an extra copy of adnAB was inserted in the chromosome. This indicates that AdnA and AdnB are both essential for Streptomyces growth. Complementation of an E. coli [delta]recB mutant by S. ambofaciens adnAB locus restored nuclease activity and cell survival in the presence or absence of DNA damaging agent, strongly suggesting that Streptomyces adnAB encodes a functional homologue of E. coli RecBCD. The key role of adnAB in homologous recombination and DNA replication is discussed. The NHEJ mechanism shows a sporadic distribution in bacteria and is known to involve the two proteins Ku and LigD. The Ku protein binds to the ends of the broken DNA and recruits the ATP-dependent ligase LigD which is a multifunctional protein carrying ligase, polymerase and sometimes nuclease activity. In silico analysis of Streptomyces genomes revealed a complex organization with a variable number of ku homologues (1 to 3) and of homologues encoding one of the three distinct LigD activities. These homologues define two conserved loci. S. ambofaciens possesses 3 ku (named kuA, kuB and kuC) and 2 ATP-dependent ligases (named ligC and ligD). Exposure to DNA damaging agents (mitomycin C, electron beam irradiation) of mutant strains got involved kuA and ligC, two conserved actors, but also variable genes such as kuC and ligD in DNA repair. These results open up new prospects to understand the role of NHEJ in the biology and genome evolution of Streptomyces.
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Lingli Zhang. Vers la compréhension des mécanismes de réparation de l'ADN chez Streptomyces : identification d'acteurs de la recombinaison. Génomique, Transcriptomique et Protéomique [q-bio.GN]. Université de Lorraine, 2014. Français. ⟨NNT : 2014LORR0104⟩. ⟨tel-01750832⟩

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