Study On PprI Phosphorylation Sites In Deinococcus Radiodurans

Deinococcus radiodurans(DR)is one of the most radioresistant bacteria,which has been used as an excellent model in DNA damage and repair mechanism.Ppr I serves as a general switch to regulate the expression of dozens of genes during the DNA repair process.Protein phosphorylation modification exists widely in prokaryotes and eukaryotes,participates in a variety of physiological activities,and significantly affects the functions of proteins.The purpose of this study is to explore the function of phosphorylation sites of Ppr I.We analyzed the function and mechanism of Ppr I phosphorylation sites through bioinformatics retrieval,mass spectrometry,molecular biology and biochemistry.The results are mainly as follows :1.Bioinformatics methods predict that there are phosphorylation,acetylation and ubiquitination in the post-translational modification of Ppr I.Ppr I is highly likely to be modified by phosphorylation,especially at the end of C-terminal,and there may be multiple phosphorylation sites in the predicted signal molecules-binding domain.2.An 18×His-tag was added in the ppr I compensatory plasmid.After enrichment of Ppr I protein and mass spectrometry measurement,12 phosphorylation sites of high abundance were obtained.These phosphorylation modification sites belong to three types: serine,threonine and tyrosine.These sites are distributed on three domains of Ppr I.3.Twelve mutant strains of Ppr I phosphorylation sites were constructed,while each site was mutated into an alanine(A)simulating continuous dephosphorylation and an aspartic acid(D)simulating continuous phosphorylation.The survival rate of 5site mutants after gamma irradiation and UV irradiation are restrained obviously,including YR2-T150 D,YR2-Y208 A,YR2-Y208 D,YR2-S302 D and YR2-T176 A,indicating that the phosphorylation site mutations have greater impact on the resistance of strains.The levels of Ddr O in the mutant strains were also detected.It was found that the expression levels of Ddr O in YR2-T150 D,YR2-Y208 D and YR2-S302 D strains remained unchanged,while the level of YR2-T176 A wasdecreased,suggesting that these point mutations disrupt the function of Ppr I,which is in accordance with the results of above resistance assay.4.Eight expression strains of Ppr I derived phosphorylation-site-mutation were constructed,including T150A/D,T176A/D,Y208A/D,S302A/D.The expression conditions of the proteins were optimized,and then purified in vitro.After that,the activity of all mutant proteins were analyzed,and showed that the mutations containing Y208 A and T176 A display significantly decreased protease activities,while the activities of the other 6 spike proteins were almost unaffected.The p ET28sf-RNAP(RNA polymarase ?)expression strain was constructed,which is used to study on the interaction between point mutation protein and RNAP.To summarize,we have found several important phosphorylation sites of Ppr I.The comprehensive analysis show that the phosphorylation of Ppr I affects its function and plays an important role in the radiation resistance of Deinococcus radiodurans.In particular,mutation of T150,T176,Y208 and S302 into alanine(A)or aspartic acid(D),significantly affected the resistance of the strains in vivo as well as the protease activities of Ppr I in vivo,indicating that these phosphorylation sites affected Ppr I-mediated repair pathway.