New mechanisms involved in the exceptional radioresistance of the bacterium Deinococcus radiodurans – Radioresistance

It is now relatively easy to genetically manipulate the D. radiodurans bacterium and its entire genome sequence has been determined. An insertion mutant library will be constructed and screened to identify new genes involved in radioresistance and/or in their regulation networks. Moreover, we are able, by adding tags to proteins, to locate them in the cells by microscopy and to follow the dynamics of their targets when they are bound to DNA.

This research project began 6 months ago. Biological tools were constructed and the feasibility of the methodologies was validated. An insertion library was constructed, new radiosensitive mutants were isolated and their characterization is now beginning. A strategy was adapted to D. radiodurans to follow the position of chromosomal loci by fluorescence microscopy.

Our project aims to better understand the properties involved in resistance to radiation and more generally to DNA damaging agents, and thus it aims to discover new approaches and ideas to improve the treatment of tumors resistant to both radiotherapy and chemotherapy.

Due to the fact that our project began 6 months ago, the timeframe is insufficient to produce publications or patents. We hope to publish the first results during the next year.

The exceptional tolerance of the bacterium Deinococcus radiodurans to very high doses of ionizing radiation is the result of several factors: very efficient repair of DNA double strand breaks, protection of proteins against oxidation and a compact structure of the nucleoid maintained after exposure to radiation. It was also proposed that prealignment of the homologous copies of the chromosomes facilitates the repair of DNA double strand breaks, but this hypothesis has not yet been tested. Analysis of D. radiodurans transcriptomes revealed a set of genes that are up-regulated in response to ionizing radiation, including several genes of unknown function specific to the Deinococcaceae. Nevertheless, the regulatory mechanisms underlying the response to radiation in the Deinococcaceae are still poorly characterized in spite of extensive work.
Our project aims
(1) to identify new genes and pathways involved in radioresistance by constructing a library of insertion mutants, using a Tn5 based mutagenesis system,. The library will be screened for radiosensitivity and the inactivated genes will be identified by arbitrary PCR followed by sequencing of the resulting PCR products. The radiosensitive mutant bacteria will be characterized for their protein protection against oxidation, their nucleoid structure, their kinetics of DNA double-strand break repair and their ability to promote massive DNA synthesis during reassembly of their genome.
(2) to elucidate the regulation network of the response to ionizing radiation by identifying and characterizing (i) the gene(s) encoding the specific binding regulator to a Radiation/Dessication Response Motif (RDRM) found in the upstream regions of radiation induced genes, taking advantage of a streptomycin selection (ii) genes involved in the regulation of the response to radiation by directly screening a D. radiodurans insertion mutant library for the impaired regulation of a reporter gene fused to an RDRM containing promoter region of a gene highly induced after irradiation.
(3) to test the prealignment of chromosomes by using the FROS (Fluorescent Repressor Operator System) methodology in D. radiodurans. To localize precise regions of each of the copies of a chromosome and to test their colocation in the nucleoid, we will construct strains containing repeats of the lacO operator in these regions and expressing the LacI repressor fused to GFP (Green Fluorescent Protein). We will target several regions of the genome and we will analyze by fluorescence microscopy the number of GFP foci per cell. This work will allow (i) to test the hypothesis of the alignment of chromosomes before and/or after irradiation (ii) to follow the choreography of the replication origin of each chromosome during the cell cycle.