DndC possesses ATP pyrophosphatase activity and is predicted to have PAPS reductase activity, while DndB is predicted to have a domain for binding Fe-S cluster proteins, as well as homology with a DNA repair ATPase and with transcription regulators 6, 9. More than half of dnd gene clusters lack dndA and contain only dndB-E, with DndA cysteine desulfurase activity functionally replaced by a host gene linked to the dndB-E cluster (for example, E. However, the functional landscape of PT modifications has not been firmly established.Īt the biochemical level, an emerging picture of Dnd protein function reveals that DndA acts as a cysteine desulfurase similar to Escherichia coli lscS and assembles DndC as a 4Fe-4 S cluster protein 16. Beginning with the original observation in Streptomyces lividans that the five-gene dnd cluster ( dndA-E) caused DNA degradation during electrophoresis 6, 7, 8, the presence of dnd genes and PT modifications has been established in >200 different bacteria and archaea, including many human pathogens 6, 9, 10, 11, 12, 13, 14, 15. We recently discovered that the dnd gene products incorporate sulphur into the DNA backbone as a PT in a sequence- and stereo-specific manner 4, 5. While the incorporation of sulphur (S) into nucleobases is well established in secondary modifications of transfer RNA (tRNA) and ribosomal RNA (rRNA) 2, the replacement of a non-bridging phosphate oxygen with sulphur as a phosphorothioate (PT) was originally developed as an artificial means to stabilize oligodeoxynucleotides against nuclease degradation 3. Secondary modifications of DNA and RNA play critical roles in cell physiology, including restriction-modification (R-M) systems in prokaryotes, and in epigenetic control of DNA replication, transcription and translation in all organisms 1. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. In contrast, PT in FF75 occurs as a single-strand modification at C psCA, again with only 14% of 160,541 sites modified. In B7A, we detect PT on both strands of G psAAC/G psTTC motifs, but with only 12% of 40,701 possible sites modified. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A.
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