Enzymatic Synthesis of Deoxyribonucleic Acid. XXXIV. Termination of Chain Growth by a 2′,3′-Dideoxyribonucleotide

2′,3′-Dideoxyribonucleoside triphosphates are analogs of the natural 2′-deoxyribonucleotide substrates of deoxyribonucleic acid polymerase but lack the 3′-hydroxyl group required for deoxyribonucleic acid chain growth. Attachment of a dideoxynucleotide blocks deoxyribonucleic acid synthesis and inhibits related reactions (pyrophosphorolysis, pyrophosphate exchange, and hydrolysis) which occur at the primer site of deoxyribonucleic acid polymerase from Escherichia coli. Attachment of a chain-terminating dideoxythymidylate group to deoxyribonucleic acid and to oligo-and polydeoxynucleotide chains is approximately a thousand times slower than that of deoxyribothymidylate. Hydrolysis and pyrophosphate exchange are inhibited to a similar extent. The requirement for a 3′-hydroxyl group for optimal rates of these reactions is discussed in terms of a model for deoxyribonucleic acid polymerase action. In the presence of an excess of polymerase, the extent of incorporation of dideoxythymidylate residues at the 3′ terminus of poly d(A-T) and of deoxyribonucleic acid chains is proportional to polynucleotide concentration, and thus permits a determination of available primer sites.