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.
Highlights
Occurring epigenetic modifications of DNA have been found prevalent in organisms from all domains of life [1]
PT modification has been found in many other bacteria and the functional study of the dnd clusters revealed the necessity of the five proteins [10]
The induced cleavage assay (ICA) assay showed that treatment of the PT-modified genomic DNA (gDNA) with iodine resulted in smaller fragment distribution compared with the control treated without iodine, whereas the gDNA from E. coli DH10B lacking PT gene cluster was not cleaved (Figure 2A)
Summary
Occurring epigenetic modifications of DNA have been found prevalent in organisms from all domains of life [1] Those modifications involve a variety of chemical groups (such as methyl group, amino acids, polyamines, monosaccharides, and disaccharides) appended to the nucleobase portion of a nucleotide. These modifications do not alter the specificity of base pairing but play an important role in protection and genetic regulation. The biochemical study of PT modification revealed that PT-modifying enzymes DndACDE function as a large protein complex [17]. It is still poorly understood how the Dnd proteins incorporate the sulfur into DNA synergistically. The tolerance of PT-modified DNA enabled the sequence identification and frequency quantitation by LC-MS/MS in the two-nucleotide sequence context after hydrolysis of the DNA samples by nucleases [3]; this method is complicated and labor-intensive
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