Structural And Biochemical Studies Of MutS2and DncA In Deinococcus Radiodurans

Deinococcus radiodurans has extremely resistance to y radiation, UV radiation, desiccation, hydrogen peroxide and other DNA damaging agents. Due to its famous oxidative stress and DNA damage repair mechanism, D. radiodurans is a popular model organism to study DNA repair. In this research, we characterized the function of two nucleases, MutS2and DncA (DR2417), in D. radiodurans.The MutS2homologues have been found widespread in most prokaryotes, which are involved in DNA repair and reactive oxygen species detoxification. The C-terminal small mutS-related (Smr) domain is critical for its endonucleolytic activity. However, the detailed catalytic mechanism is still unclear. In this study, we first investigated the in vivo role of drMutS2protein in Deinococcus radiodurans. mutS2and recA mutS2double knockout mutants were constructed because the phenotype was strongly masked by the predominant homologous recombination DNA repair pathway in this bacterium. Compared with the recA mutant, cells devoid of both genes showed increased sensitivity to ionizing radiation and oxidative agents, suggesting that drMutS2is involved in RecA-independent mechanisms that enhance cellular resistance to oxidative stress-induced DNA damage. Moreover, the basal level of reductase activity and thiamine biosynthesis was induced in the absence of mutS2. To characterize its catalytic residues, the Smr domain was crystallized and soaked in buffer containing manganese ions. In contrast to native crystals, the space group of manganese-derivative crystals transformed from monoclinic to orthorhombic unexpectedly. This type of crystals showed improved diffraction resolution to1.2A, which has the highest resolution of currently known Smr structures. Structural comparison revealed that three acidic amino-acid residues, which are all located in the al helix, changed the rotamer states after metal soaking. Mutational analysis of conserved residue glutamic acid710to alanine yielded a drMutS2variant with impaired nuclease activity, and could only partially rescue the radiosensitive phenotype of the mutS2null strain, indicating that glutamic acid710is the catalytic residue.It was reported that DncA (DR2417), which belonged to the β-CASP family in D. radiodurans, showed an Mn2+dependent3’-5’exonuclease efficiently on dsDNA and ssDNA/dsDNA junction endonuclease activities while a very low level activity on RNA, and the protein was purified under denaturing conditions. In this study, we expressed and purified the natural DncA protein. The DncA protein showed an Mn2+dependent5’-3’exonuclease activity on ssDNA rather than dsDNA, and possessed overhang processing endonucleolytic activity. Meanwhile, DncA could open hairpin DNAs like Artemis at a lower pH at6.5. Different from the previous data, DncA showed strongly RNase activity. The crystal structure of DncA apo contained the β-lactamase domain, β-CASP domain and C-terminal domain just like RNase J. A catalytically inactive mutant D175A was abtained and used to cocrystalize with ssDNA. The structure of the complex showed an enlarged pocket for substrates, which indicated that DncA could bind to more complex DNA. The C-terminal of DncA was required for dimerization, and the C-terminal truncated protein showed almost null activity on complex DNA such as hairpin DNA. Genetics study found that both dr2417and the C-terminal truncated domain (dr2417△C) could not be deleted completely in D. radiodurans. The phenotypes of dr2417△C were more obviously than R1and△dr2417following by y radiation and other DNA damage agents. Above all, DncA and its C-terminal were important for D. radiodurans.