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.