Genomic Mapping Of Phosphorothioation And The Convergence Of DNA Methylation And DNA Phosphorothioation In Bacteria

In 1952,Alfred Hershey,cooperated with Martha Chase,conducted phage infection experiment confirming the genetic material comes from the DNA not proteins.Combining with the earlier result from Avery-MacLeod-McCarty experiment,the idea believed by the biologists that proteins were the genetic material has been subverted.This experiment applied isotope labelling,in which 35S for the protein while 32P for the DNA due to the sulfur element was believed to be exclusive in protein.However,in recent years,sulfur has been found in the backbones of DNA,which is called phosphorothioation(PT),from bacteria and archaea.Further studying showed this novel modification were mediated by five proteins,DndABCDE,transporting the sulfur atom from L-cysteine to the DNA backbone replacing the non-bridging oxygen.Usually,the PT modification is paired with its restriction enzymes,DndFGH,to form a restriction-modification(RM)system for resisting the foreign DNA while keeping the stability of own genetic material.Based on high performance liquid chromatography conbined with mass spectrum,the scientists have done qualitative and quantitative analysis for PT modification in d(XPSY)(X=A,T,C or G,Y= A,T,C or G)level from different bacteria and found the sequence as well as the frequency of PT modification was bacteria-dependent.However,the longer motif of PT modification is not easy to found due to the limitation of techbology.To be more important,the genomic location of PT modification is a mystery for a long time.The answer for this scientific issue might be helpful for excavation of the biological function of PT modification.In this study,we successfully mapped PT modifications on the genome from Vibrio cyclitrophicus FF75 by appling the third generation sequencing technology,single molecular real time(SMRT)sequencing technology.We found,in FF75,PT modification occurs in 5'-CPSCA-3' motif showing a strong bias against T and a moderate preference towards A or G.Only 14%of the possible site have been PT modified and a single-strand modification has been presented in FF75.With this genomic PT map,we found PT modification was random as well as evenly distributed acrossing the genome,and the modification situation in individual bacteria showed heterogeneity which indicated partial and dynamic feature.Besides,the author has found the PT-related restriction-modification process could be interference with DNA methylation-associated restriction-modification system.On the one hand,the phosphorothioated modification on the DNA backbones could share the same DNA sequence with DNA methylation to generate the hybrid modified structure d(GPS6mA)which showed the same RP configuration compared with solo PT modification.Nevertheless,the PT modified oligos would reduce the activity of Dam methylase in vitro.Unexpectedly,in this study we found DndFGH was temperature-sensitive and the transcription of the restriction gene cluster,dndFGH,would be up-regulated at a relatively lower temperature.Following this clue,we found the low-temperture restriction phenomenon could be blocked by the methylated DNA,which means DNA methylation could play the same role as DNA phosphorothioation in protecting the genome from restricted by DndFGH.