Functional Analysis Of DrMoaE And DrDciA In Response To DNA Damage Stress In D.radiodurans

The DNA damage resistance of organisms is mainly derived from the dual collaboration of DNA damage defense and repair.Deinococcus radiodurans has been used as one of the model organisms to study the mechanism of DNA damage defense and repair due to its special extreme resistance ability,and thus the study of its mechanism will provide important insights into the DNA damage resistance of organisms.Antioxidant mechanisms and DNA replication are important in DNA damage defense and repair.Moa E and Dci A are involved in the oxidation-related Moco（molybdenum cofactor）biosynthesis and DNA replication,respectively,but their roles in DNA damage resistance of D.radiodurans are not yet clear.Therefore,this study investigates the mechanism of resistance of Dr Moa E and Dr Dci A to DNA damage stress in D.radiodurans.Firstly,Dr Moa E was purified by in vitro purification,optimized to obtain protein crystals and structurally resolved.The Dr Moa E is structurally shown as a dimer with conservative four-stranded antiparallelβ-sheets and an anionic-substrate binding pocket consisting of His109,Arg110,and Arg45,with Arg110 being the key residue for dimerization formation.Knockout of the drmoa E,an essential gene for Moco biosynthesis,causes a reduced growth rate and shows sensitive resistance to H₂O₂,high doses of UV andγ-ray.Meanwhile,drmoa E transcript levels increase under oxidative stress,and its deletion results in a significant accumulation of ROS in D.radiodurans in vivo.However,the decrease in superoxide dismutase,as well as catalase activity,is not significant in theΔdrmoa E mutant strain.However,the activity of DMSO reductase containing Moco is lost and DMSO-mediated electron transfer from the respiratory chain is restricted.Transcript levels of genes encoding Moco-containing related enzymes are increased in wild-type strains after H₂O₂ treatment.The base oxidative damage analog 6-hydroxyaminopurine（HAP）causes decreased survival and increased mutation frequency inΔdrmoa E.The results suggest that Dr Moa E may metabolize DNA oxidative damage products in response to DNA damage defense.By structure prediction analysis,the Dci A structural domain consists of 3β-sheets and 4α-helices,but the folding pattern varies in different species.There is relative independence between the N-terminal domain（Dci A domain）and the C-terminal domain（zinc finger domain,and the C-terminal unknown functional domain）of Dr Dci A.Biochemical analysis shows that the DNA binding ability of Dr Dci A is derived from the C-terminal domain.Δdrdci A and each structural domain heterozygous mutant strains have slower growth rates,and their resistance to UV andγ-ray stress is reduced,but the resistance of each structural domain mutant strain is higher than that ofΔdrdci A under radiation stress.In addition,Δdrdci A mutant strains show an increased spontaneous mutation rate.Dr Dci A is localized in the cytoplasm under physiological conditions,but concentrated in the nuclear region under UV stress,while the transcript levels of drdci A increase 8-fold.Secondly,the replicative helicase Dr Dna B of D.radiodurans interacts with Dr Dci A,and ATPase activity and helicase activity of Dr Dna B are triggered by Dna G^HBD.Meanwhile,Dr Dci A prefers to bind the substrate ds DNA of the replication fork structure,which not only enhances the ATPase activity of triggered Dr Dna B,but also reduces the unwinding time from 60 minutes to about 40minutes.These suggest that Dr Dci A is involved in DNA damage repair,accelerating replication unwinding.In summary,this study provides a detailed functional and structural analysis of the Dr Moa E and Dr Dci A and explores that they deal with DNA damage stress by affecting the DNA damage defense and repair of D.radiodurans,respectively.The results not only broaden the functional knowledge of Dr Moa E and Dr Dci A,but also provide an important addition to the insight into the mechanism of DNA damage resistance in organisms.