Structure of the nucleoïd and radioresistance of Deinococcus radiodurans and Deinococcus deserti – DEINOCOCCUS

Deinococcus radiodurans and Deinococcus deserti (the latter recently isolated from desert sand) belong to a family of bacteria characterized by an exceptional capacity to cope with the lethal effects of DNA-damaging agents, including ionizing radiation, UV light and desiccation. The radioresistance of these bacteria is linked to their extraordinary ability to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments, whereas the genome of most organisms is irreversibly shattered under the same conditions. The genome sequence of D. radiodurans was published in 1999 and we recently obtained that of D. deserti (Septembre 2006). A typical set of prokaryotic repair genes has been identified in their genomes but the molecular mechanisms for their extraordinary radioresistance are not yet fully understood. Active (DNA repair processes), and passive mechanisms (nucleoid organization) are probably intimately combined to enable survival from stresses. Different DNA repair pathways were proposed for the reconstruction of an entire genome from DNA fragments in D. radiodurans: extended synthesis-dependent strand annealing (ESDSA), homologous recombination (HR) and non-homologous end joining (NHEJ). The tightly packed structure of the nucleoid of radioresistant bacteria may play an important role in these processes by limiting diffusion of the DNA fragments and holding together free DNA ends. D. radiodurans cells adopt a condensed ring-like nucleoid structure that remains unaltered after high-dose gamma-irradiation whereas the nucleoid structure of D. deserti does not adopt a fixed shape suggesting that strong nucleoid condensation, rather than the shape of the nucleoid, may be the common trait among radioresistant organisms. In the present project, different strategies will be used to better understand the respective roles of active (DNA repair processes) and passive (nucleoid organization) mechanisms enabling resistance to DNA damaging agents in Deinococcus : (1) A detailed comparison of D. radiodurans and D. deserti is currently being done both at the genome and proteome levels. This will be an invaluable basis to select a priori for novel key candidate proteins involved in the radiation resistance mechanisms. (2) We will construct different test strains to evaluate in vivo the respective contribution of ESDSA, HR and NHEJ processes in DNA double strand break repair. In particular, we will adapt to D. radiodurans a strategy developed in Saccharomyces cerevisiae by introducing HO-induced DNA double-strand breaks (DSB) in a dispensable gene and we will test DSB repair and its fidelity in different mutant strains. From these data, the involvement of different candidate proteins in the DSB repair pathways will be established. (3) Proteins often function as components of multisubunit complexes or transiently interact with partners. To decipher protein-protein interaction networks involved in radioresistance, we will purify protein complexes formed in vivo in D. radiodurans using the Sequencial Peptide Affinity method (SPA-tag). Proteins present in the complexes will be identified by mass spectrometry and their role in DNA repair will be determined. (4 We will characterize the mechanisms of compaction of the Deinococcus nucleoid and investigate the role of the highly condensed structure of the nucleoid in Deinococcus radioresistance. For this, we will isolate the nucleoid-associated proteins in D. radiodurans and D. deserti and characterize the nucleoid-associated proteins using genetic and biochemical approaches