Development of Methods Derived from Iodine-Induced Specific Cleavage for Identification and Quantitation of DNA Phosphorothioate Modifications
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biomolecules-10-01491.pdf
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1.86 MB
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Checksum (MD5)
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Author(s)
Zhu, Sucheng
Zheng, Tao
Kong, Lingxin
Li, Jinli
Cao, Bo
DeMott, Michael S.
Sun, Yihua
Chen, Ying
Deng, Zixin
Dedon, Peter C.
Date Issued
October 28, 2020
Publisher
Multidisciplinary Digital Publishing Institute
Citation
Biomolecules 10 (11): 1491 (2020)
Version
Final published version
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Center for Environmental Health Sciences
Singapore-MIT Alliance in Research and Technology (SMART)
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DOI of Published Version
https://doi.org/10.3390/biom10111491
