Quantitative mapping of DNA phosphorothioatome reveals phosphorothioate heterogeneity of low modification frequency.
PLOS GENETICS(2019)
摘要
Phosphorothioate (PT) modifications of the DNA backbone, widespread in prokaryotes, are first identified in bacterial enteropathogens Escherichia coli B7A more than a decade ago. However, methods for high resolution mapping of PT modification level are still lacking. Here, we developed the PT-IC-seq technique, based on iodine-induced selective cleavage at PT sites and high-throughput next generation sequencing, as a mean to quantitatively characterizing the genomic landscape of PT modifications. Using PT-IC-seq we foud that most PT sites are partially modified at a lower PT frequency (< 5%) in E. coli B7A and Salmonella enterica serovar Cerro 87, and both show a heterogeneity pattern of PT modification similar to those of the typical methylation modification. Combining the iodine-induced cleavage and absolute quantification by droplet digital PCR, we developed the PT-IC-ddPCR technique to further measure the PT modification level. Consistent with the PT-IC-seq measurements, PT-IC-ddPCR analysis confirmed the lower PT frequency in E. coli B7A. Our study has demonstrated the heterogeneity of PT modification in the bacterial population and we also established general tools for rigorous mapping and characterization of PT modification events at whole genome level. We describe to our knowledge the first genome-wide quantitative characterization of PT landscape and provides appropriate strategies for further functional studies of PT modification. Author summary Phosphorothioate (PT) modification is a novel DNA modification, previous studies showed that PT modifications in E. coli occure at G(ps)AAC/G(ps)TTC motifs, but the modification frequency at each site are not known. In this study, we introduced two methods: PT-IC-seq, which could quantitatively characterize the genomic landscape of PT modifications; and PT-IC-ddPCR, which could measure PT modification frequency precisely. Through these two new methods, heterogeneity, an important feature of PT modification, is revealed intuitively and accurately. The modifications revealed in this study provide a basis for the final revelation of the physiological functions of PT and give valuable reference for other DNA modification studies.
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关键词
dna phosphorothioatome
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