Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

Significance We have solved crystal structures of ternary Thermus thermophilus Argonaute (Ago) complexes with guide and target DNA in cleavage-incompatible, cleavage-compatible, and postcleavage states in the 2.2- to 2.3-Å resolution range, thereby identifying the relative positions of catalytic residues, a pair of Mg 2+ cations, and the nucleophilic water poised for in-line attack on the cleavable phosphate. These higher resolution structures represent snapshots of distinct key steps in the catalytic RNase H-mediated cleavage pathway, providing additional detailed insights into Ago-mediated cleavage chemistry of target strands. Importantly, a Glu residue shifts from an “outside” to an “inside” conformation where it inserts into the catalytic pocket to complete a catalytic tetrad during the transition from a cleavage-incompatible to a cleavage-compatible conformation.