Study On The Extracellular Nuclease In Deinococcus Radiodurans

Extracellular DNA molecules are present in most environments where organisms live. These DNA molecules can be reused as a nutrient after degradation, or promote the organic evolution by horizontal gene transfer (HGT) after absorbed into cells. Extracellular nuclease plays an important role in regulating the amount of extracellular DNA molecules. Deinococcus radiodurans is so extremely resistant to ionizing radiation, UV radiation, hydrogen peroxide, desiccation and other chemical agents that it is a model organism for the research of DNA damage and repair. D. radiodurans cells release large oligo-nucleotides (～1kb) into the surrounding medium immediately after γ-radiation, and the extracellular nuclease DRB0067rapidly degrades these oligo-nucleotides to nucleotides to prevent the reincorporation of damaged bases into the genome and protect the organism from excessive mutagenesis. In the current thesis, the cell growth and survival of D. radiodurans after extracellular DNA or dNMPs treatment were investigated, and the new biological function and significance of DRB0067protein in D. radiodurans were described. The main results are as follows:1. High concentrations of extracellular DNA fragments inhibited the growth of D. radiodurans and D. radiopugnans, but had no effect on the other five organisms, such as E. coli and so on. The same mass concentration of dNMPs also had no effect on the gowth of D. radiodurans and E. coli. These results suggest that high concentrations of extracellular DNA molecules threaten the growth of D. radiodurans and should be rapidly degraded to nucleotides to avoid it.2. Extracellular dGMP enhanced D. radiodurans tolerance to oxidative stress, such as H2O2and γ-radiation, but had no effect on E. coli. The same mass concentration of extracellular DNA fragments had no effect on both D. radiodurans and E. coli. Further experiments indicated that extracellular dGMP stimulates the activity of one catalase (KatA, DR1998), and induced gene transcription including the extracellular nuclease gene drb0067. 3. DRB0067, the only extracellular nuclease secreted by D. radiodurans, had the endonuclease and the3’â†'5’ exonuclease activity, and could degrade plamid, ssDNA and dsDNA. This protein is secreted through Sec pathway, and modulated by y-radiation, indicating that DRB0067participates in the resistance of ionizing radiation in D. radiodurans.4. We constructed a drb0067knockout strain(Δdrb0067), and found that Δdrb0067grew more slowly in the presence of DNA fragments, had a lower transformation, and became more sensitive to H2O2and γ-radiation compared with wild type. The survival fraction tests of extracellular dGMP and Oligo(dG)50on Δdrb0067tolerance to H2O2indicated that D. radiodurans can use dGMP, a DNA component, to enhance its tolerance to oxidative stress.In summary, the degradation of extracellular DNA into dNMPs by extracellular nuclease DRB0067serves many purposes in D. radiodurans, including converting extracellular DNA into nutrients, thus reversing cell growth inhibition and protecting the organism from excessive mutagenesis, and finally enhancing D. radiodurans tolerance to oxidative stress.