SgrAI is a sequence specific DNA endonuclease that functions through an unusual enzymatic mechanism that is allosterically activated 200-500 fold by effector DNA, with a concomitant expansion of its DNA sequence specificity. Using single-particle transmission electron microscopy to reconstruct distinct populations of SgrAI oligomers, we show that, in the presence of allosteric, activating DNA, the enzyme forms regular, repeating helical structures that are characterized by the addition of DNA-binding dimeric SgrAI subunits in a run-on manner. We also present the structure of oligomeric SgrAI at 8.6 Å resolution, demonstrating a novel conformational state of SgrAI in its activated form. Activated and oligomeric SgrAI displays key protein-protein interactions near the helix axis between its N-termini, as well as allosteric protein-DNA interactions that are required for enzymatic activation. The hybrid approach reveals an unusual mechanism of enzyme activation that explains SgrAI’s oligomerization and allosteric behavior.

SgrAI is a type II restriction endonuclease that cleaves an unusually long recognition sequence and exhibits allosteric self‐modulation of DNA cleavage and sequence specificity. We have shown that SgrAI forms very large aggregates (High Molecular Weight Species, HMWS) in the presence of sufficient concentrations of SgrAI and primary site DNA, that are dependent on the concentration of the DNA bound SgrAI dimer. We propose that the oligomers of DNA bound SgrAI (ie. HMWS) are the activated form of the enzyme. The unusual biochemical activity of SgrAI may have evolved to sequester activated SgrAI endonuclease on invading phage DNA and away from genomic DNA. We present new data showing that an uncleavable (3′S modified) primary site DNA activates SgrAI, therefore providing conclusive evidence that the primary site need not be cleaved by SgrAI in order to induce the activated conformation. Finally, while we have solved several crystal structures of SgrAI bound to DNA, including one showing two DNA bound SgrAI dimers interacting in a domain swapped tetramer, the structure of the activated oligomeric state has remained elusive. We present here new structural information gleaned from electron microscopy as well as ion mobility mass spectrometry (IM‐MS). The new data show that SgrAI/DNA oligomers form regular, rod shaped structures, with the DNA bound SgrAI dimer as the basic building block. IM‐MS collision crosssections are consistent with the rod‐like morphology of the oligomer seen in the electron microscopy, and a model of the oligomer based on SgrAI/DNA crystal structures has been proposed.

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