A DNA phosphorothioation pathway via adenylated intermediate modulates Tdp machinery.

In prokaryotes, the non-bridging oxygen in the DNA sugar-phosphate backbone can be enzymatically replaced by a sulfur atom, resulting in phosphorothioate (PT) modification. However, the mechanism underlying the oxygen-to-sulfur substitution remains enigmatic. In this study, we discovered a hypercompact DNA phosphorothioation system, TdpABC, in extreme thermophiles.

Single-molecule optical mapping of the distribution of DNA phosphorothioate epigenetics.

DNA phosphorothioate (PT) modifications, with the nonbridging phosphate oxygen replaced by sulfur, governed by DndABCDE or SspABCD, are widely distributed in prokaryotes and have a highly unusual feature of occupying only a small portion of available consensus sequences in a genome. Despite the presence of plentiful non-PT-protected consensuses, DNA PT modification is still employed as a recognition tag by the restriction cognate, for example, DndFGH or SspE, to discriminate and destroy PT-lacking foreign DNA. This raises a fundamental question about how PT modifications are distributed along DNA molecules to keep the restriction components in check.

SspABCD-SspE is a phosphorothioation-sensing bacterial defence system with broad anti-phage activities.

Bacteria have evolved diverse mechanisms to fend off predation by bacteriophages. We previously identified the Dnd system, which uses DndABCDE to insert sulfur into the DNA backbone as a double-stranded phosphorothioate (PT) modification, and DndFGH, a restriction component. Here, we describe an unusual SspABCD-SspE PT system in Vibrio cyclitrophicus, Escherichia coli and Streptomyces yokosukanensis, which has distinct genetic organization, biochemical functions and phenotypic behaviour.