An in vitro DNA phosphorothioate modification reaction

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.

Microbial Arsenal of Antiviral Defenses – Part I

“…Besides the presence of PglZ, the common feature of all BREX systems is the presence of ATPase and methyl transferase. In the Type IV systems, the latter is replaced by a PAPS reductase, an enzyme that can be involved in DNA phosphorothioation [192]. The most prevalent is the Type I BREX and systems of this type have been experimentally investigated in B. subtilis, E. coli, and V. cholerae (where it has been found in the SXT conjuga tive elements) [191,193,194].…”

“…The PT modification occurs as a result of the activi ty of the system of dndABCDE genes (Dnd is a phenotype associated with DNA degradation), which encode the DndA cysteine desulfurase, the DndC PAPS reductase, the DndD ATPase/nicking endonuclease, and a small protein DndE that binds nicked DNA, while DndB regu lates transcription of the dndBCDE operon and deter mines proportion of the PT modified sites in the genome [204, 207 209]. Not all stages of the biochemical pathway involved in PT modification have been determined but it is known that cysteine serves as a donor of the sulfur atom that is transferred to DndC and next incorporated in an energy dependent manner into the DNA that was pre liminary nicked at specific sites by DndD [192,210,211]. Recently, it was shown that the dnd genes could also be BIOCHEMISTRY (Moscow) Vol.…”

Involvement of the DNA Phosphorothioation System in TorR Binding and Anaerobic TMAO Respiration in Salmonella enterica

“…The contradiction between the in vivo and in vitro results prompted us to construct a mutant strain, HY1, expressing the chromosomal dndBCDE operon in which a single cysteine residue (C280) was replaced with a serine residue in DndC. The single-point C280S mutation completely abolished the DNA PT modification ( 35 ), but the complete DndCDE protein complex was retained. Interestingly, despite the loss of the DNA PT modification, torC in the HY1 strain displayed the same transcription level as that in wild-type Cerro 87 ( Fig.…”

“…IscS (a DndA homolog), DndC, DndD, and DndE have been observed to form a complex at an approximate molecular ratio of 1:1:1:1 in vitro ( 34 ). A recent study showed that DndCDE-IscS confers the DNA PT modification in the presence of SAM, Mg 2+ , ATP, and cysteine, supporting the PT reaction through a radical SAM mechanism ( 35 ). All these observations suggest that sulfur is first transferred from cysteine to DndA and then to the cysteine residues in DndC before it is ultimately incorporated into the DNA backbone through the coordinated functions of DndD and DndE ( 36 ).…”

DNA Phosphorothioate Modifications Are Widely Distributed in the Human Microbiome

“…Recently, another PT-modifying gene cluster has been reported, sspA-D , which confers cells with 5′-C ps CA-3′ on the single strand [ 4 ]. The DndA protein, which can be functionally substituted by an IscS (a cysteine desulfurase located elsewhere in the genome) [ 5 , 6 ], transfers sulfur into the Fe-S cluster of DndC [ 5 , 7 ]. SspA and SspD may share the same initial sulfur mobilization pathway with DndA and DndC, respectively [ 4 ].…”