Phosphorothioate (PT) modification of DNA, with sulfur replacing a nonbridging phosphate oxygen, was recently discovered as a product of the dnd genes found in bacteria and archaea. Given our limited understanding of the biological function of PT modifications, including sequence context, genomic frequencies, and relationships to the diversity of dnd gene clusters, we undertook a quantitative study of PT modifications in prokaryotic genomes using a liquid chromatography-coupled tandem quadrupole mass spectrometry approach. The results revealed a diversity of unique PT sequence contexts and three discrete genomic frequencies in a wide range of bacteria. Metagenomic analyses of PT modifications revealed unique ecological distributions, and a phylogenetic comparison of dnd genes and PT sequence contexts strongly supports the horizontal transfer of dnd genes. These results are consistent with the involvement of PT modifications in a type of restriction-modification system with wide distribution in prokaryotes.DNA modification | bioanalytical chemistry | sulfur P hosphorothioate (PT) modification of DNA, in which sulfur replaces a nonbridging phosphate oxygen, was originally developed as an artificial means to stabilize oligodeoxynucleotides against nuclease degradation (1). However, we recently discovered that the dnd gene products incorporate sulfur into the DNA backbone as a PT in a sequence-and stereo-specific manner (2). Beginning with the original observation in Streptomyces lividans 1326 that the five-gene dnd cluster (dndA-E) caused DNA degradation during electrophoresis (3), the presence of dnd genes has been established in dozens of different bacteria and archaea (4). An emerging picture of Dnd protein function reveals that DndA acts as a cysteine desulfurase and assembles DndC as a 4Fe-4S cluster protein (5). DndC possesses ATP pyrophosphatase activity and is predicted to have PAPS reductase activity, whereas DndB has homology to a group of transcriptional regulators (4, 6). A DndD homologue in Pseudomonas fluorescens Pf0-1, SpfD, has ATPase activity possibly related to DNA structure alteration or nicking during PT incorporation (7).This progress in defining the biochemistry of PT modifications belies a lack of understanding of the biological function of PT modifications, such as the variety of sequence contexts, the distribution of modifications across prokaryotic genomes, and the relationship of PT sequence contexts to the diversity of known dnd gene clusters (4). We have approached this problem with a highly quantitative study of PT modifications in prokaryotic genomes using a liquid chromatography-coupled tandem quadrupole mass spectrometry (LC-MS/MS) approach. The results reveal a diversity of quantized PT sequence contexts consistent with a role for PT modifications as part of a restrictionmodification system. Results and DiscussionDevelopment of a Sensitive Method to Quantify PT Modifications in Bacterial Genomes. We approached the problem of defining the biological function of PT modifications by q...

The chemical diversity of physiological DNA modifications has expanded with the identification of phosphorothioate (PT) modification in which the nonbridging oxygen in the sugar-phosphate backbone of DNA is replaced by sulfur. Together with DndFGH as cognate restriction enzymes, DNA PT modification, which is catalyzed by the DndABCDE proteins, functions as a bacterial restriction-modification (R-M) system that protects cells against invading foreign DNA. However, the occurrence of systems across a large number of bacterial genomes and their functions other than R-M are poorly understood. Here, a genomic survey revealed the prevalence of bacterial systems: 1,349 bacterial systems were observed to occur sporadically across diverse phylogenetic groups, and nearly half of these occur in the form of a solitary gene cluster that lacks the restriction counterparts. A phylogenetic analysis of 734 complete PT R-M pairs revealed the coevolution of M and R components, despite the observation that several PT R-M pairs appeared to be assembled from M and R parts acquired from distantly related organisms. Concurrent epigenomic analysis, transcriptome analysis, and metabolome characterization showed that a solitary PT modification contributed to the overall cellular redox state, the loss of which perturbed the cellular redox balance and induced to reconfigure its metabolism to fend off oxidative stress. An in vitro transcriptional assay revealed altered transcriptional efficiency in the presence of PT DNA modification, implicating its function in epigenetic regulation. These data suggest the versatility of PT in addition to its involvement in R-M protection.

Topographical guidance and chemotaxis are crucial factors for peripheral nerve regeneration. This study describes the preparation of highly aligned poly(ε-caprolactone) (PCL) fiber conduits coated with a concentration gradient of nerve growth factor (NGF) (A/G-PCL) using a newly designed electrospinning receiving device. The A/G-PCL conduits are confirmed in vitro to enhance and attract the neurite longitudinal growth of dorsal root ganglion (DRG) neurons toward their high-concentration gradient side. In vivo, the A/G-PCL conduits are observed to direct a longitudinal stronger attraction of axons and migration of Schwann cells in 15 mm rat sciatic nerve defects. At 12 weeks, rats transplanted with A/G-PCL conduits show satisfactory morphological and functional improvements in g-ratio, total number, and area of myelinated nerve fibers as well as the sciatic function index, compound muscle action potentials, and muscle wet weight ratio as compared to aligned PCL fibers conduits with uniform NGF (A/U-PCL). The performance of A/G-PCL is similar to that of autografts. Moreover, mRNA-seq and RT-PCR results reveal that Rap1, MAPK, and cell adhesion molecules signaling pathways are closely associated with axon chemotactic response and attraction. Altogether, by combining structural guidance with axon chemotaxis, the NGF-gradient/ aligned PCL fiber conduits represent a promising approach for peripheral nerve defect repair.