Terminal deoxynucleotidyl transferase indiscriminately incorporates ribonucleotides and deoxyribonucleotides

Terminal deoxynucleotidyl transferase (TdT) catalyzes the condensation of d eoxyribonucleotides on 3 ' -hydroxyl ends of DNA strands in a template-inde pendent manner and adds N-regions to gene segment junctions during V(D)J re combination. Although TdT is able to incorporate a few ribonucleotides in v itro, TdT discrimination between ribo- and deoxyribonucleotides has never b een studied. We found that TdT shows only a minor preference for incorporat ion of deoxyribonucleotides over ribonucleotides on DNA strands. However, i ncorporation of ribonucleotides alone or in the presence of deoxyribonucleo tides generally leads to premature chain termination, reflecting an impeded accommodation of ribo- or mixed ribo/deoxyribonucleic acid substrates by T dT. An essential catalytic aspartate in TdT was identified, which is a firs t step toward understanding the apparent lack of sugar discrimination by Td T.