Study On A New Type Of DNA Phosphorothioation-based Antiviral System In Archaea

The DNA phosphorothioation(PT)modification refers to the DNA modification form in which a non-bridging oxygen on the DNA phosphodiester bond is replaced by a sulfur.It is the first time to clarify the sixth element "sulfur" on DNA.In previous studies,the scientists found that DNA PT modification is controlled by proteins encoded by dndABCDE gene cluster.DNA PT modification is widespread and quantized in bacterial genomes and has various physiological functions.However,up to now,the research on PT modification has been carried out in bacteria.It is the first time to identify DNA PT modification in archaea.As an important branch of the three-domain evolutionary tree of life,it is particularly necessary to study DNA PT modification in archaea.In this study,firstly retrieve the dndCDEA gene clusters of some archaea through bioinformatics analysis,and identify the PT modification and modification frequency of five strains of halophilic archaea,the N.bangense JCM10635,H.jeotgali A29,H.amylolyticum CGMCC 1.10121,H.limi JCM16811 and H.salinum JCM19729 by analysis of LC-MS/MS,core sequence is(5'-GATC-3').SMRT technology was used to precisely locate the PT modification in four of the archaea.In this study,the dnd gene cluster in archaea H.jeotgali A29 was further heterogeneously expressed,and it was found that dndCDEA gene cluster and the nearby pbeABCD gene cluster together constitute a new antiviral system.We further carried out segment deletion experiments to identify the essential genes of the system,and carried out point mutation experiments at key sites to determine that the antiviral function of the system depends on PT modifications phenotypes.In addition,since the core sequence of PT modification of this system is 5'-GATC-3',which is the same as the methylation modification mediated by Dam methyltransferase,the relationship between antiviral system based on DNA PT modification and methylation modification in archaea was preliminarily explored in this study.This study is devoted to the exploration of DNA PT modification system in archaea,breaking through the bottleneck of the research on DNA PT modification which is limited to bacteria,and expanding the understanding of the physiological significance of DNA PT modification as an apparent modification,enriching our understanding of the restriction modification system.