GAMMATOLERANS

Thermococcus gammatolerans was isolated from a deep-sea hydrothermal vent and was shown to be one of the most radioresistant organisms known amongst the Archaea isolated so far. It withstands a 30 kGy pulse radiolysis dose without any detectable lethality. To gain insights into the mechanisms involved in the high radiotolerance of this extraordinary biological model, we started an ambitious research project. We sequenced and annotated its genome. We analyzed its proteome from exponential- and stationary-phase cells by an extra-large shotgun proteomic approach. We performed transcriptome kinetics over the time required for T. gammatolerans to restore an intact chromosome after gamma-irradiation of exponentially growing cells. We are now characterizing the role of the proteins involved in DNA repair and DNA damages tolerance in T. gammatolerans. For this, we defined four highly complementary tasks: 1) establishing the proteome dynamics in T. gammatolerans cells in response to gamma-irradiation and pulse radiolysis, 2) describing the role of several key transcription factors involved in cell recovery and defining their regulons by a SELEX-adapted strategy, 3) identifying proteins interacting directly with DNA damages by a ligand fishing method (chemical proteomics) and, 4) studying with biochemical approaches at least 5 key DNA repair proteins and DNA-binding proteins, chosen from our transcriptomic data and the ligand fishing results. By means of label-free quantitative shotgun proteomics, we will quantify the most abundant proteins present in the cells in a time-course analysis after gamma-irradiation (2,5 and 5,0 kGy doses) and pulse-radiolysis (5,0 and 30 kGy doses). Their level of post-translational modifications (oxidation and phosphorylation states) will be established. Comparative analysis along the kinetics should give novel insights into the physiological differences depending on the nature and amount of stress. We have already selected ten T. gammatolerans transcription regulators that are differentially expressed during cell recovery after irradiation. We started their heterologous production and already purified four of them. Using a SELEX-inspired strategy, we presently define the DNA consensus pattern recognized by these regulators and their targets on the genome. DNA footprinting, EMSA and other biochemical methods will further define the DNA-protein interactions and the characteristics of these key regulators. With specialized chemists, we are developing DNA probes mimicking gamma-irradiation damages that will be used as baits to trap proteins recognizing these damages out of extracts prepared from T. gammatolerans stressed cells. This innovative chemical proteomics approach should point at new proteins recognizing DNA damages, as well as their co-purified partners. Finally, we will characterize at least five key proteins involved in DNA repair or radiotolerance pointed by our transcriptomic and proteomic approaches. We will analyze their structural and enzymatic characteristics in terms of DNA repair and DNA binding. An invaluable insight into T. gammatolerans radiotolerance is at hand, with the probable discovery of really novel mechanisms for DNA repair in the Archaea-Eukarya lineage. Based on our first preliminary assays, our massive proteomic investment should be really fruitful in pointing the key proteins involved in DNA repair and radiotolerance. Moreover, this project will be a nice opportunity to develop new knowledge regarding the integration of multi-OMICs data and specific regulatory mechanisms in hyperthermophilic organisms.