Structural and biochemical characterization of a novel DinG containing an endonuclease domain.

DinG-like proteins are members of the XPD-family SF2 helicases and are widely distributed in bacteria, yet they exhibit remarkable diversity in domain architecture and biological function. A distinct subgroup of DinG homologs harboring an N-terminal PD-(D/E)XK-type nuclease domain, designated EndoDinGs, has remained uncharacterized. Here, we report the first structural and functional characterization of this subgroup using Finegoldia magna DinG (FmaDinG) as a representative example. FmaDinG displays Mn2+-preferred endonuclease activity together with Mg2+-dependent 5'-3' unidirectional helicase activity. We determined the crystal structure of the FmaDinG-ssDNA-ADPNP complex at 2.26 Å resolution. Structural and biochemical analyses reveal a direct role of the Arch domain in DNA engagement and suggest a gating mechanism within the nuclease domain that regulates DNA entry, a feature that may be specific to the EndoDinG subgroup. Key residues required for nuclease and helicase activities were identified by site-directed mutagenesis. Together, these results support a mechanistic model in which EndoDinGs such as FmaDinG function as coordinated nuclease-helicase machines for DNA processing.