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In the reaction catalyzed by the polynueleotide-joining enzyme from Escherichia coti, phosphodiester bonds are synthesized between the 3'-hydroxyl and 5'phosphoryl termini of properly aligned DNA chains coupled to the cleavage of the pyrophosphate bond of DPN. 1-4 The first step in this over-all reaction occurs in the absence of DNA and consists of the formation of a covalently linked enzymeadenylate (E-AMP) intermediate and simnultaneous release of NMN5I I (Fig. 1).

Enzymatic joining of polynucleotides, V. A DNA-adenylate intermediate in the polynucleotide-joining reaction.