July 7, 2019  |  

Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences.

Authors: Cao, Bo and Chen, Chao and DeMott, Michael S and Cheng, Qiuxiang and Clark, Tyson A and Xiong, Xiaolin and Zheng, Xiaoqing and Butty, Vincent and Levine, Stuart S and Yuan, George and Boitano, Matthew and Luong, Khai and Song, Yi and Zhou, Xiufen and Deng, Zixin and Turner, Stephen W and Korlach, Jonas and You, Delin and Wang, Lianrong and Chen, Shi and Dedon, Peter C

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. However, bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. 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). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. 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. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.

Journal: Nature communications
DOI: 10.1038/ncomms4951
Year: 2014

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