The Regulatory Mechanism Of DndB On DNA Phosphorothioation And The Interaction Of DndB With Other Dnd Proteins

DNA phosphorothioation(PT)is the first reported physiological modification on the sugar-phosphate backbone,in which the non-bridging oxygen is replaced by sulfur in a sequence-selective and R_P stereo-specific manner.Widespread in bacteria and archaea,PT systems have undergone extensive horizontal gene transfer.The nuclease resistance as well as the redox and nucleophilic properties of PT sulfur make PT modification a versatile player in restriction-modification(R-M)defense,epigenetic regulation,environmental fitness and the maintenance of cellular redox homeostasis.The physiological abundance of PT modification is strictly controlled and its biosynthesis involves the five Dnd ABCDE proteins:Dnd ACDE catalyzes the oxygen-sulfur swap on the DNA backbone;as a repressor of the dnd BCDE operon,DndB can impede the transcription of dnd CDE and its own genes by special attachment to the promoter region upstream of the dnd cluster,thereby negatively controlling the cellular PT levels.However,whether the DndB-promoter interaction is regulated by certain unknown environmental or cellular stimulus(effector),together with the DndB responsive mechanism involved in PT regulation by the potential effector,still remains unknown.To this end,we firstly conducted an amino acid sequence analysis and found a strictly conserved DGQHR motif within DndB protein.Site-directed mutagenesis evaluation showed that the DGQHR motif is critical to the PT regulatory function of DndB,but does not directly participate in the DndB-promoter interaction,which pointed to the mechanism of effector-mediated DndB regulation.Subsequently,guided by protein homology modeling and associated sequence conservation analysis,a colorimetric Pi release assay showed that native DndB readily hydrolyzes ATP at?32 nmol Pi/min/mg,and the DGQHR-mediated PT alterations result from its impact on DndB ATPase activity.By means of enzyme kinetic assay,electrophoretic mobility shift assay(EMSA)and protein oligomerization state analysis(BN-PAGE),we next unveiled the key role that the ATPase property of DndB plays in the tight control of PT modification:as a repressor,DndB recognizes ATP as an“inducer”effector to modulate the expression of dnd CDE genes and itself;the ATP-binding event likely causes conformational changes in DndB and disturbs its interaction with the promoter DNA,which triggers the dissociation of DndB-ATP from the promoter region for allowance of transcriptional initiation;the ATP hydrolysis activity resets DndB-ATP back to free DndB that recovers its DNA-binding conformation to interact with the promoter again,leading to transcriptional re-inhibition of the dnd BCDE operon.Finally,physiological experiments using arsenate or bleomycin treatment further revealed that DndB could sense the rise or decline of cellular ATP levels to correspondingly up-regulate or down-regulate the expression of PT-modifying genes.Taken together,the repressor protein DndB,serving as an ATP sensor,is able to modulate the transcription of dnd BCDE operon and the resulting PT levels in response to the cellular ATP concentrations in an unusual auto-regulated and reusable manner.This study discovered that tight control of PT modification is mediated via the dual attributes of DndB protein as not only a transcriptional repressor but also an ATPase.On the one hand,DndB senses cellular ATP levels to determine genome-wide PT modification frequencies,which offers an economical way to fine-tune the ATP-consuming process of PT sulfur incorporation.To our knowledge,it is the first reported ATP-sensing regulation case among DNA epigenetic modification system.On the other hand,the presented DndB features of auto-regulation and reusability allow strict control of Dnd-modifying proteins and the resultant cellular PT levels,which supplies theoretical support for the widespread distribution of PT system as a restriction-modification(R-M)defense barrier after horizontal gene transfer.Moreover,this ATP-modulated strict control of PT modification also renders its frequency suitable for its other diverse physiological functions.It has been reported that bacterial ATP concentrations change significantly when the cells come into different growth phases or face various environmental stresses(UV radiation,bleomycin,inorganic arsenate,sharp alterations in temperature/osmotic pressure/p H).According to our results,these pronounced changes in ATP concentration,theoretically,could be sensed by DndB to modulate the expression of dnd BCDE genes and,possibly,cellular PT levels.It thus may be hypothesized but must be experimentally verified that the PT changes following ATP variations play more roles in the adaptation and multifaceted functions of cells.Therefore,the presented mechanism of ATP-sensing and tight modulation on the genomic PT modification by auto-regulated and reusable DndB provides new insight into PT physiology and functionality.In the other part of this dissertation,GST Pull-down technology was adopted to establish a systematic analysis of the interactions between PT-modifying proteins in pf0-1.Further,natural Dnd CDE complex was prepared by protein co-expression and co-purification.This part of work lays a foundation for future study of PT modification on its biochemical mechanism,in vitro reconstruction,and directed biosynthesis.