[DNA modification by sulfur: mechanistic insights drives synthetic biotechnologies forward]

As the genetic material of living organisms, DNA contains diverse chemical modifications beyond the four bases. Since the first discovery of DNA methylation a century ago, over 17 natural DNA modifications have been identified, including 5-methylcytosine (5mC), N6-methyladenosine (6mA), N4-methylcytosine (4mC), and 5-hydroxymethylcytosine (5hmC). These modifications typically do not affect base pairing but may modulate DNA-protein interactions, thereby playing critical roles in physiological processes and disease occurrence. Early studies predominantly focused on base modifications, while the discovery of DNA sulfur modification marked a breakthrough-the first natural modification involving a new element (sulfur) replacing the non-bridging oxygen species in the DNA phosphodiester bond backbone, forming a phosphorothioate (PT) linkage. Recent studies have elucidated the genomic distribution, sequence context, and biological functions of PT modifications. This review highlights the bacterial defense systems associated with PT modifications, their molecular recognition mechanisms, and emerging applications as enabling technologies in gene editing, nucleic acid detection, and bacteriophage-resistant industrial strain development, providing insights for synthetic biology.