Investigating the interactions between DNA and DndE during DNA phosphorothioation

Abstract

The DNA phosphorothioate modification is a novel physiological variation in bacteria. DndE controls this modification by binding to dsDNA via a mechanism that remains unclear. Structural analysis of the wild‐type DndE tetramer suggests that a positively charged region in its center is important for DNA binding. In the present study, we replaced residues G21 and G24 in this region with lysines, which increases the DNA binding affinity but does not affect the DNA degradation phenotype. Structural analysis of the mutant indicates that it forms a new tetrameric conformation and that DndE interacts with DNA as a monomer rather than as a tetramer. A structural model of the DndE–DNA complex, based on its structural homolog P22 Arc repressor, indicates that two flexible loops in DndE are determinants of DNA binding

Structural and Functional Analysis of DndE Involved in DNA Phosphorothioation in the Haloalkaliphilic Archaea Natronorubrum bangense JCM10635

“…Wild-type E. coli B7A DndE adopts a four-leaf clover-like tetrameric conformation by hydrogen bonds between the side chain of K20 in each monomer with G21 and/or G24 in the next monomer, generating a positively charged hole at the center of the tetramer, which is involved in DNA binding ( 25 ). When the positively charged region was expanded by the introduction of K21 and K24, the resultant DndE G21/24K variant exhibited increased DNA binding affinity ( 26 ). Sequence alignment of DndE from bacterial E. coli B7A and archaeal N. bangense JCM10635 showed only 22% identity ( Fig.…”