In Vivo Mutational Characterization Of DndE In DNA Phosphorothioate Modification And The Establishment Of The Purification System Of Dnd Proteins

When carrying out experiments of electrophoresis of Streptomyces lividans genomic DNA, scientists found that the bands in the gel were not clear but presented the state of degradation and dispersion. The phenomenon later was named DNA degradation (Dnd phenomenon).After in-depth analysis of this phenomenon, it was found that the Tris oxidized derivatives produced in anode in electrophoresis would cause cleavages in the double chains of bacterial chromosome DNA, which led to the degradation of bacterial DNA. Further investigation of this phenomenon reveals that this mechanism is related to dnd gene cluster. And this gene cluster which was located in bacterial chromosome DNA can lead to the replacement of non-bridging oxygen atoms for sulfur atoms in the DNA backbone, resulting in DNA modification with the characteristics of specific RP spatial structure and selected sites. This finding reveals the sixth element of life-sulfur. Subsequent studies reported that this modification was completely different from methylation and widely distributed in a series of prokaryotes.Earlier researches had predicted some functions of each gene in the dnd gene cluster by homology analysis, measuring the enzymatic activity of the purified protein in vitro, or analyzing its function and active site through protein crystallization. After with crystal structure analysis and fluorescence polarization of DndE protein, Wei Hu et al. had reported the replacement of six positively charged residue sites in the DndE surface (including K17, K18, K20, K53, K87and K91) by alanine showed significant decreases in binding affinities to specific dsDNA. All these variants showed much weaker or non-detectable binding to nicked sdsDNA, suggesting that all these positively charged residues might be important for interaction with nicked dsDNA. The study in this dissertation was just based on the above report. We carried out research on DndE protein from S. enterica serovar Cerro87to study its biological function in vivo by LC-MS/MS tandem mass spectrometry and site-directed mutagenesis. We also expect to get the purified proteins associated with the modification system in S. enterica serovar Cerro87to reconstruct the PT process in vitro and elucidate the biological function of each protein. Based on that, the biochemical pathway in the modification may be revealed clearly.pJTU1238is a plasmid containing dndBDCE, the complete modification gene cluster of S. enterica serovar Cerro87, which can generate phosphorothioation phenomenon by heterologous expression. This research firstly carried out site-directed mutations and in-frame deletion of dndE gene on plasmid pJTU1238, obtaining a series of pJTU1238derivative plasmids, respectively introduced into a PT-deficient mutant of S. enterica serovar Cerro87, XTG102. And then we introduced the plasmid pJTU1238and its derivative plasmids into a series of Escherichia coli type strains, such as DH10B, DH5a, ER2796and Mach Ⅰto study the influence of mutated dndE gene on phosphorothioate modification frequency and site-selectivity. Different expression hosts containing pJTU1238or its derivative plasmids had different phosphorothioate modification frequency.Results showed that the two lysine sites K18and K20on the DndE protein were indeed the key sites which influenced the function of DndE protein through a series of experiments. But mutation at both loci at the same time did not affect the occurrence of phosphorothioate modification but only reduce the modification frequency.Besides the role of combining the band gap of double stranded DNA in vivo, we assumed that DndE protein might also play some other unknown functions in phosphorothioate modification system. Then the experiments of qRT-PCR and Western blot were carried out to study the influence of mutated dndE gene on its transcriptional and translational level. And the influence of mutated dndE gene on phosphorothioate modification under the same transcriptional level and translational level was also analyzed. It was found that different mutated sites of dndE gene in the same bacterial host affected greatly different on modification frequency whereas the same mutated site in different bacteria also functioned disparately/variouly.At present, there are only a few reports of protein purification or heterologous expression of each gene of phosphorothioation cluster in S. enterica serovar Cerro87. We have performed protein expression and purification of dndA, dndC, dndD and dndE from S. enterica serovar Cerro87so as to understand the function of each gene and the biochemical pathway of phosphorothioation further by reconstructing this modification system with these purified Dnd proteins in vitro.