Structural And Functional Studies On Sspa Of The Type ⅡDNA Phosphorothioate Modification System In Streptomyces Clavuligerus

DNA phosphorothiation is the first confirmed physiological modification occurred on the DNA backbone with sequence-selective and stero-specific(Rp)manner,in which sulfur atom is substituted with non-bridging oxygen governed by five proteins encoded by dndABCDE gene cluster.At present,DNA phosphorothiate system only exists in prokaryotes and contains two types,type I and II.Streptomyces lividans,the first strain used in the research field,is the typical representative of type Ⅰ,double-stranded modification on DNA conferred by the aforementioned five genes.Type II system,governed by SspABCD gene cluster,confers single-standed modification with high frequency and was found in recent years,such as V.cyclitrophicus FF75.Long-term research found that restriction cognate dndFGH,located downstream of dndBCDE,and modifier gene form RM system with special regualtion to prevent host from invasion of foreign DNA without phosphorothiation.The exploration of type II modification system is ongoing.In this study,we detected the Streptomyces clavuligerus ATCC 27064 possessed type II phosphorothiate modification-d(CPSC)Rp by analysis of LC-MS/MS.Two experiments of gene blast and heterologous expression identified four genes sspABCD which is responsible for PT modification.On the basis of SspA crystal,combining the result of homologous protein sequences alignment and SspA structural analysis,we found that three motifs are extremely conserved on the SspA surface.Three variants of full mutation and a series of mutations about the three motifs abolished PT modification or decreased the quantity,confirming that sspA is critical for DNA phosphorothiate pathway.The result of protein structural comparison indicated that SspA has a nuclease function similar with FokI restriction endonuclease and nicking endonuclease N.BspD6I.Studise in vitro showed that SspA non-specifically cut spercoiled DNA in the presence of most divalent ions,especially Mg2+ and Mn2+,while Zn2+ and high concentration of Cu2+ obviously inhibited the activity.Using linear DNA and oligonucleotide DNA as substrates to the study,the same results were observed to further confirm the non-specific nuclease activity of the protein.The mutation of E18 with negative,ion binding site by analysis of co-structure with divalent Mn2+,severly affected cleavage activity,further supporting the necessity of divalent metal ions on protein nuclease activity.Besides,the morphology of SspA in aqueous solution is the form of monomer and its crystal structure shows the dimer state,which is similar to FokI protein.Key sites Y103/F24 and E204,identified by analysis of main force of SspA dimer formation,were substituted by amino acid residues with oppsite charge.We found the two variants protein completely loss the cleavage activity.In this study,the sequence analysis of S.clavuligerus ATCC 27064 was carried out by SMRT sequencing method to provided important basis for the precise localization of phosphorothiate modification in genome.Finally,the study on the interaction between type II proteins,provides a strong theoretical basis for the subsequent biochemical pathway exploration of phosphorothiate modification.The phenomena of the interaction between SspB and SspC,SspB and SspD was observed,which may need other experiments to further confirm it.This study is devoted to the exploration on type II phosphorothiation to lay a solid foundation for the formation mechanism of the new DNA phosphorothiate modification.