A specific cytidine-cytidine-adenosine (CCA) sequence is required at the 3'-terminus of all functional tRNAs. This sequence is added during tRNA maturation or repair by tRNA nucleotidyltransferase enzymes. While most eukaryotes have a single enzyme responsible for CCA addition, some bacteria have separate CC- and A-adding activities. The fungus, Schizosaccharomyces pombe, has two genes (cca1 and cca2) that are thought, based on predicted amino acid sequences, to encode tRNA nucleotidyltransferases. Here, we show that both genes together are required to complement a Saccharomyces cerevisiae strain bearing a null mutation in the single gene encoding its tRNA nucleotidyltransferase. Using enzyme assays we show further that the purified S. pombe cca1 gene product specifically adds two cytidine residues to a tRNA substrate lacking this sequence while the cca2 gene product specifically adds the terminal adenosine residue thereby completing the CCA sequence. These data indicate that S. pombe represents the first eukaryote known to have separate CC- and A-adding activities for tRNA maturation and repair. In addition, we propose that a novel structural change in a tRNA nucleotidyltransferase is responsible for defining a CC-adding enzyme. (C) 2018 Elsevier Inc. All rights reserved.