An in vitro DNA phosphorothioate modification reaction
Authors: Pu, T., Mei, Z., Zhang, W., Liang, W.J., Zhou, X., Liang, J., Deng, Z. and Wang, Z.
Journal: Molecular Microbiology
Volume: 113
Issue: 2
Pages: 452-463
eISSN: 1365-2958
ISSN: 0950-382X
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 Escherichia 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. Adenosine triphosphate (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.
https://eprints.bournemouth.ac.uk/33110/
Source: Scopus
An in vitro DNA phosphorothioate modification reaction.
Authors: Pu, T., Mei, Z., Zhang, W., Liang, W.-J., Zhou, X., Liang, J., Deng, Z. and Wang, Z.
Journal: Mol Microbiol
Volume: 113
Issue: 2
Pages: 452-463
eISSN: 1365-2958
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 Escherichia 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. Adenosine triphosphate (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.
https://eprints.bournemouth.ac.uk/33110/
Source: PubMed
An in vitro DNA phosphorothioate modification reaction.
Authors: Pu, T., Mei, Z., Zhang, W., Liang, W.-J., Zhou, X., Liang, J., Deng, Z. and Wang, Z.
Journal: Molecular microbiology
Volume: 113
Issue: 2
Pages: 452-463
eISSN: 1365-2958
ISSN: 0950-382X
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 Escherichia 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. Adenosine triphosphate (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.
https://eprints.bournemouth.ac.uk/33110/
Source: Europe PubMed Central
An in vitro DNA Phosphorothioate Modification Reaction.
Authors: Pu, T., Mei, Z., Zhang, W., Liang, W.-J., Zhou, X., Liang, J., Deng, Z. and Wang, Z.
Journal: Molecular Microbiology
Volume: 113
Issue: 2
Pages: 452-463
ISSN: 0950-382X
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.
https://eprints.bournemouth.ac.uk/33110/
Source: BURO EPrints