In vitro substrate specificities of 3′–5′ polymerases correlate with biological outcomes of tRNA 5′-editing reactions

•Mitochondrial tRNA in many protozoan eukaryotes undergo 5′-editing.•TLPs use 3′–5′ polymerase activity to repair 5′-truncated editing substrates.•A. castellanii TLP2 tolerates G–U pairs in editing substrates better than other TLPs.•TLP activities correlate with biological outcomes for G–U base pairs during editing.•Biochemical evidence suggests specialized functions for different eukaryotic TLPs.

Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5′-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G–U base pairs, wherein some species remove G–U base pairs during 5′-editing, while others retain G–U pairs in the edited tRNA. We tested whether 3′–5′ polymerases that catalyze the repair step of 5′-editing exhibit organism-specific preferences that explain the treatment of G–U base pairs. Biochemical and kinetic approaches revealed that a 3′–5′ polymerase from Acanthamoeba castellanii tolerates G–U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing.