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Development of Methods Derived from Iodine-Induced Specific Cleavage for Identification and Quantitation of DNA Phosphorothioate Modifications

1
State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China
2
College of Life Sciences, Qufu Normal University, Qufu 273165, Shandong, China
3
Department of Biological Engineering and Center for Environmental Health Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
4
Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, Singapore 138602, Singapore
*
Author to whom correspondence should be addressed.
Biomolecules 2020, 10(11), 1491; https://doi.org/10.3390/biom10111491
Received: 30 September 2020 / Revised: 23 October 2020 / Accepted: 26 October 2020 / Published: 28 October 2020
(This article belongs to the Section Molecular Biology)
DNA phosphorothioate (PT) modification is a novel modification that occurs on the DNA backbone, which refers to a non-bridging phosphate oxygen replaced by sulfur. This exclusive DNA modification widely distributes in bacteria but has not been found in eukaryotes to date. PT modification renders DNA nuclease tolerance and serves as a constitute element of bacterial restriction–modification (R–M) defensive system and more biological functions are awaiting exploration. Identification and quantification of the bacterial PT modifications are thus critical to better understanding their biological functions. This work describes three detailed methods derived from iodine-induced specific cleavage-an iodine-induced cleavage assay (ICA), a deep sequencing of iodine-induced cleavage at PT site (ICDS) and an iodine-induced cleavage PT sequencing (PT-IC-Seq)-for the investigation of PT modifications. Using these approaches, we have identified the presence of PT modifications and quantized the frequency of PT modifications in bacteria. These characterizations contributed to the high-resolution genomic mapping of PT modifications, in which the distribution of PT modification sites on the genome was marked accurately and the frequency of the specific modified sites was reliably obtained. Here, we provide time-saving and less labor-consuming methods for both of qualitative and quantitative analysis of genomic PT modifications. The application of these methodologies will offer great potential for better understanding the biology of the PT modifications and open the door to future further systematical study. View Full-Text
Keywords: DNA; phosphorothioate (PT) modifications; iodine-induced cleavage (ICA); ICDS; PT-IC-Seq DNA; phosphorothioate (PT) modifications; iodine-induced cleavage (ICA); ICDS; PT-IC-Seq
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Zhu, S.; Zheng, T.; Kong, L.; Li, J.; Cao, B.; DeMott, M.S.; Sun, Y.; Chen, Y.; Deng, Z.; Dedon, P.C.; You, D. Development of Methods Derived from Iodine-Induced Specific Cleavage for Identification and Quantitation of DNA Phosphorothioate Modifications. Biomolecules 2020, 10, 1491.

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