Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease

Filament or run-on oligomer formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms run-on oligomeric (ROO) filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the 3.5 [A] cryo-electron microscopy structure of the ROO filament of SgrAI bound to a mimic of cleaved primary site DNA and Mg2+. Large conformational changes stabilize a second metal ion cofactor binding site within the catalytic pocket and facilitate assembling a higher-order enzyme form that is competent for rapid DNA cleavage. The structural changes illuminate the mechanistic origin of hyper-accelerated DNA cleavage activity within the filamentous SgrAI form. An analysis of the protein-DNA interface and the stacking of individual nucleotides reveals how indirect DNA readout within filamentous SgrAI enables recognition of substantially more nucleotide sequences than its low-activity form, thereby expanding DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAIs catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.