Two-metal ion mechanism of DNA cleavage by activated fi lamentous SgrAI

Enzymes that form fi lamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric fi laments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI fi lamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to fi lamentous SgrAI maximize contacts between different copies of the enzyme within the fi lament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of fi lamentous SgrAI solved using cryo-EM. The fi rst structure, resolved to 3.3 angstrom, is of fi lamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 angstrom, is of WT fi lamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of fi lamentation induced conformational changes.