Function Of Dndei Involved In Dna Phosphorthioation And The Phosphorthioate Modification Frequency

Phosphorothioation?PT?is a novel DNA modification,in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur.It is a stereospecific post-replicative modification and regarded as the first-described physiological modification on the DNA backbone.The physiological PT modification is widespread in bacteria and occurs in diverse sequence contexts in different bacterial genomes,implying a significant impact on bacteria.PT modifications are governed by a large family of five-gene clusters termed as the dndA-E.Functional study of the five genes has been performed but how sulfur incorporated into DNA is still obscure.To better understand the biochemical mechanism of PT modification,functional analysis of a novel PT-modifying enzyme?DndEi?from Riemerella anatipestifer was performed in this study.DndEi is a hybrid protein composed of an E domain that is homologous to DndE and a putative DNA helicase domain.In vitro study indicates that DndEi presents an ATP-dependent DNA helicase activity.Deletion of the helicase domain in vivo leads to DNA phosphorothioation deficiency,suggesting an essential role of helicase activity in PT modification.Subsequent analysis reveals that DndEi has an ATPase activity strongly stimulated by double-stranded DNA.Intriguingly,DNA substrates containing a GAAC/GTTC motif stimulate the ATPase activity of DndEi more effectively than DNA fragments without this motif and DNA fragments containing a phosphorothioated G_psAAC/G_psTTC motif.These data suggest that DNA helicase is indispensable for PT modification and the ATP consumption is elevated when oxygen is replaced by sulfur in the PT modification process.Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences,but the modification frequency is unknown.For precise and quantitative phosphorothioation analysis,we developed a method for modification frequency determination in this study.DNA samples were first treated with iodine.PT-modified DNA molecules can be specifically cleaved at the modified phosphodiester linkage so that the modified and unmodified DNA molecules can be distinguished.The accurate modification frequency was then calculated by quantifying the total DNA and the unmodified DNA molecules by droplet digital PCR.This method was used for determination of the modification frequency in Escherichia coli B7A.The modification frequencies of eight loci on genomic DNA were less than 50%,indicating an incomplete modification.Modification frequencies of cells at different growing phase were determined and the frequencies of some loci changed in the growing cycle,implying that PT modification is dynamic.