# Molecular Insight into the Recognition of DNA by the DndCDE Complex in DNA Phosphorothioation

## TL;DR

This study reveals how a bacterial protein complex recognizes DNA to modify it with sulfur atoms, helping to defend against foreign DNA invasion.

## Contribution

The study provides a structural and functional model of the DndCDE complex and its DNA recognition mechanism through protein structure prediction and biochemical validation.

## Key findings

- DndC's 'specificity loop' is crucial for recognizing the DNA sequence targeted for phosphorothioation.
- DndD has a conserved structural surface that likely mediates dimerization and complex assembly.
- The DndCDE complex undergoes dynamic transitions between open and closed states to bind and release DNA.

## Abstract

In a vast variety of prokaryotes such as Escherichia coli and Streptomyces lividans, the DNA degradation (Dnd) CDE protein complex (consisting of DndC, DndD, and DndE), together with the DndA/IscS protein and the DndFGH complex, function as a defense barrier to prevent the invasion of non-self-DNA. The DndCDE complex introduces phosphorothioation (PT) modifications into DNA, and the DndFGH complex specifically cleaves non-PT DNA and, thus, restricts horizontal gene transfer and phage invasion. Despite the central importance of the DndCDE complex in DNA PT modification, which catalyzes the oxygen–sulfur swap on DNA, our understanding of this key complex remains poor. Here, we employed protein structure prediction to provide a reasonably reliable prediction of the structure of the DndCDE complex and a 23 bp DNA-DndCDE complex. We found that among the three proteins in the DndCDE complex, DndC, especially its “specificity loop”, plays a key role in recognizing the consensus PT modification sequence. In addition, the DndD protein is found to possess a highly conserved structural surface on its globular domain, presumably mediating the dimerization of DndD as well as the DndCDE complex. Furthermore, our normal mode analysis showed that there exists a dynamic transition between a closed and an open state for the DndCDE complex, facilitating its association and release of DNA. Our conclusions were corroborated by biochemical assays using purified proteins. On the whole, we provide molecular insights into the assembly and DNA-recognition mechanism of a central protein complex involved in DNA phosphorothioation.