Study On The DNA Damage Response Gene DdrI In Deinococcus Radiodurans

Deinococcus radiodurans R1 is one of the most radioresistant bacteria on the earth. This particular species can stand a wide range of DNA-damaging agents such as gamma-radiation, UV, H2O2 and desiccation. For many years, it has been an ideal model organism to investigate the integrity and stability of the genome. However, the underlying molecular mechanisms remain poorly understood. System singaling the stresses from eniverment are required in all three kindom of lifes. Phosphorylation on histidine and aspirate are the main signal-transduction pathways in prokaryote, named two-compenents system, which consists of a kinase and a response regulator. Previous studies of our laboratory showed that DR2418, a key participator of the radio-resistance of Deinococcus radiodurans, binds to the promoter region of dr0997, encoding of cAMP receptor protein (CRP). Proteins of CRP family are very important to Deinococcus radiodurans, and regulate more than 200 genes. Therefore, we focused on this gene in this study.1. The gene dr0997 and its homologue gene dr1646 were successfully knocked out with a kanamycin-resistance gene cassette. We found that the growth of both knockout strains were remarkablely delayed, indicating both genes are required for the metabolism.2. We investigated the survival of the mutant strainsΔDR0997 andΔDR1646 under stresses of DNA-damaging agents. We found that both mutants are sensitive to gamma-radiation, indicating that both genes may be involved in the recovery of D. radiodurans from radiation damage. However, absence of DR0997 only caused a very modest reduction of survival capability when cells were treated UV radiation or H2O2.3. Then, DNA microarray was used to analyze the alteration of transcription profiles when DR0997 is abscent.3033 genes were detected in the microarray, taking up of 95.26% in all predicted ORF. We found that 79 genes were significantly down-regulated, while 28 genes were significantly up-regulated in the mutant compared to these in the wildtype strain. Many of these genes are involved in the synthesis of NADPH and acetyl coenzyme A. These suggest that DR0997 may be involved in the regulation of these pathways.4. To further investigate the biological function of DR0997 protein, the recombinant DR0997 protein was induced and purified here. The gene dr0997 was cloned and resequenced. It has been comfirmed that the ORF of this gene consists of only 612 bp, which is smaller than the predicted one in the Genebank. We also successfully constructed the strain for recombinant DR0997 protein expression and purified the recombinant protein.Taken together, the gene dr0997 plays an important role in the radioresistance of Deinococcus radiodurans Rl, and regulates the carbohydrate metabolism. The work to reveal the underlying mechanisms is proceeding.