Discovery And Identification Of DNA Repair Proteins In Deinococcus Radiodurans By Proteomics

Deinococcus radiodurans (DR) is a Gram-positive, red-pigmented, nonmotile and nonphathaogenous bacterium that was originally identified as a contaminant of irradiated canned meat. The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans Rl is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284,156 base pairs.DR is best known for its extreme resistance to the lethal effects of ionizing radiation. Exponential phase cultures of the DR Rl strain survive exposure to gamma radiation at doses as high as 5,000 Gy without loss of viability or evidence of DNA damage induced mutation. 6000 Gy of irradiation will induce approximately 200 DNA double-strand breaks in DR chromosomes, but it is still able to reconstruct a functional genome from chromosomal fragments without any mutation. Over the past few years, although many factors have been put forward to explain this phenomenon, and even though the complete genomic DNA sequence of D. radiodurans is now known, the molecular mechanism underlying this phenotype is still poorly understood.In order to reveal the mechanisms of extreme radioresistance and DNA repair in Deinococcus radiodurans, we examined proteome changes in a wild type strain following y-irradiation using 2-dimensional polyacrylamide gel electrophoresis and silver staining. Proteins were identified with peptide mass fingerprinting using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) after tryptic in-gel digestion. These proteins exhibited various cellular functions, Most of them have not been reported to be relevant to radioresistance.In DR genome, there are about 50 percent proteins which functions are still unknown. Thus clarifying the functions of these proteins may help us to better understanding the mechanisms of extreme radioresistance in this organism. In our results, there are a few hypothetical proteins whose expression level increased or induced recovering from ionizing radiation. Two hypothetical proteins were disrupted successfully, and the survivals of both disruptants were decreased dramatically. The results of present paper suggest that it is feasible to discovery novel radioresistance-associated proteins by proteomics methods.