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An in vitro DNA Phosphorothioate Modification Reaction.

Pu, T., Mei, Z., Zhang, W., Liang, W.-J., Zhou, X., Liang, J.., Deng, Z. and Wang, Z., 2020. An in vitro DNA Phosphorothioate Modification Reaction. Molecular Microbiology, 113 (2), 452-463.

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DOI: 10.1111/mmi.14430

Abstract

Phosphorothioation (PT) involves the replacement of a nonbridging phosphate oxygen on the DNA backbone with sulfur. In bacteria, the procedure is both sequence- and stereo-specific. We reconstituted the PT reaction using purified DndCDE from Salmonella enterica and IscS from E. coli. We determined that the in vitro process of PT was oxygen-sensitive. Only one strand on a double-stranded (ds) DNA substrate was modified in the reaction. The modification was dominant between G and A in the GAAC/GTTC conserved sequence. The modification between G and T required the presence of PT between G and A on the opposite strand. Cysteine, S-adenosyl methionine (SAM), and the formation of an iron-sulfur cluster in DndCDE (DndCDE-FeS) were essential for the process. Results from SAM cleavage reactions support the supposition that PT is a radical SAM reaction. ATP promoted the reaction but was not essential. The data and conclusions presented suggest that the PT reaction in bacteria involves three steps. The first step is the binding of DndCDE-FeS to DNA and searching for the modification sequence, possibly with the help of ATP. Cysteine locks DndCDE-FeS to the modification site with an appropriate protein conformation. SAM triggers the radical SAM reaction to complete the oxygen-sulfur swapping.

Item Type:Article
ISSN:0950-382X
Additional Information:Funding Information the National Key Research and Development Program of China. Grant Number: 2018YFA0900702 the Ministry of Science and Technology. Grant Number: 973 program, 2015CB554203 the National Science Foundation of China. Grant Numbers: 21661140002, 31470830, 91753123
Uncontrolled Keywords:S-adenosyl methionine (SAM) ; cysteine ; iron-sulfur cluster ; phosphorothioate modification ; radical SAM reaction ; sulfur incorporation
Group:Faculty of Science & Technology
ID Code:33110
Deposited By: Unnamed user with email symplectic@symplectic
Deposited On:03 Dec 2019 13:02
Last Modified:31 Mar 2020 14:55

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