Structural basis of sequence-specific cytosine deamination by double-stranded DNA deaminase toxin DddA

Abstract

Abstract An interbacterial deaminase toxin DddA catalyzes cytosine-to-uracil conversion in double-stranded (ds) DNA and enables CRISPR-free mitochondrial base editing, but the molecular mechanisms underlying its unique substrate selectivity remained unknown. Here we report crystal structures of DddA bound to a dsDNA substrate containing the 5'-TC target motif. The structures show that DddA binds to the minor groove of a sharply bent dsDNA and engages the target cytosine extruded from the double-helix. DddA Phe1375 intercalates in dsDNA and displaces the 5' (–1) thymine, which in turn replaces the target (0) cytosine and forms a non-canonical T-G base-pair with the juxtaposed guanine. This “domino effect” mechanism allows DddA to locate the target cytosine without flipping it into the active site. Biochemical experiments show that DNA base-mismatches enhance DddA deaminase activity and relax its sequence selectivity. Based on the structural information, we further identified DddA mutants that exhibit attenuated activity or altered substrate preference. Our studies may help design novel tools useful in genome editing or other applications.