Structure of a Bacterial DNA Phosphorothioation Complex
EMSL Project ID
51264
Abstract
Phosphorothioation is a DNA modification that replaces a phosphate oxygen atom with a sulfur in the DNA backbone. It is a common artificial DNA modification in synthesized oligonucleotides, because it increases stability and decreases immunogenicity of oligonucleotides in cells. Though it was only known as an artificial DNA modification, researchers found this modification is also naturally present in genomic DNA of many bacterial and several archaeal species. DNA phosphorothioation appears to work in a restriction-modification system or as a response to oxidative stress. This phenotype is associated with a five gene cluster, dndABCDE, and the protein products DndA, DndC, DndD, and DndE form a complex capable of modifying DNA with phosphorothioates in a sequence-dependent manner. From previous work, DndA is a cysteine desulfurase that transfers a sulfur from cysteine to an iron-sulfur cluster (it is sometimes replaced by another bacterial homologue IscS in several species); DndC is an iron-sulfur cluster protein that is proposed to directly add the sulfur to a phosphate through an unknown ATP-dependent mechanism; DndD is an ATP-dependent protein that is thought to nick DNA or stabilize its secondary structure; and DndE has been shown to be a nicked DNA-binding protein. In addition, other work has shown the sequence specificities of this complex in different bacterial species by high-throughput sequencing and a rough map of the intersubunit interactions through biochemical experiments. Nevertheless, there is insufficient structural or biochemical data to illustrate precisely how this complex catalyzes the phosphorothioation reaction.To address this question, the goal of this project is to determine the structure of the DndCDE complex found in a strain of E. coli by cryo-electron microscopy. Cryo-EM is well-suited to studying this large >440 kilodalton complex with possible structural dynamics. A high-resolution cryo-EM structure of this complex, accompanied by biochemical experiments, will elucidate key mechanistic details of the phosphorothioation reaction. Besides the catalytic mechanism, a structure will illustrate the complex's contribution to bacterial fitness in a restriction-modification system and in oxidative stress conditions. Phosphorothioation has been hypothesized to enable pathogenic bacteria to resist reactive oxygen species released by human phagocytes, suggesting this complex could be a novel antibiotic drug target specific to pathogenic bacteria. In addition, structural information would allow guided engineering of this complex for gene- editing applications.
Thus far, we have purified the DndCDE complex from a strain of E. coli to high homogeneity and taken negative-stain micrographs that show distinct 10 nm particles. We have not yet examined frozen grids with this complex in low dose conditions, so we would like to screen grids of the DndCDE complex on the Talos Arctica microscope in a day session to optimize sample and freezing conditions that yield high quality particles. If a suitable grid is found and time permits, we would like to obtain a preliminary overnight dataset to guide further cryo-EM and biochemical experiments.
Project Details
Start Date
2020-02-15
End Date
2020-08-15
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members