A recent communication' described an activity in Escherichia coli extracts which converts hydrogen-bonded circles of X phage DNA to covalently closed circles. In this paper we describe a rapid assay for joining of DNA strands, again based on closure at the ends of X DNA, and utilize it to obtain a purification and characterization of an enzyme catalyzing this activity, the "DNA-joining enzyme." The joining reaction has the biochemically intriguing feature of utilizing diphosphopyridine nucleotide (DPN) in a nonoxidative manner as the cofactor in phosphodiester bond formation. The reaction requires a 5'-phosphoryl terminus on the DNA strand that, in the presence of the 3'-terminus of a suitably juxtaposed hydrogen-bonded DNA strand, is incorporated into a 3'-5' phosphodiester bond. In this process, DPN is cleaved to 5'-A1VJP and nicotinamide mononucleotide (NMN). Joining of polydeoxyribonucleotide strands through 3'-5' phosphodiester bonds by an E. coli enzyme, with possibly similar cofactor requirement, has lately also been reported by Olivera and Lehman.2 An ATP-dependelt enzyme with related activity from T4-infected cells has been briefly described by Weiss, Live, and Richardson,3 and Becker, Gefter, and Hurwitz.4 Experimental Procedure.-Materials: The following commercial products were used: E. coli alkaline phosphatase, snake venom phosphodiesterase, spleen phosphodiesterase, pancreatic DNase, and micrococcal nuclease (Worthington); alcohol dehydrogenase (Calbiochem); DPN, DPNH, TPN, TPNH, thionicotinamide-DPN, coenzyme A (P-L Biochemicals); a-DPN, 3acetylpyridine-DPN, desamino-DPN, NMN, ADP-ribose, FMN, FAD, sRNA (E. coli), and nucleoside mono-, di-, and triphosphates (Calbiochem); calf thymus DNA (Worthington). Glucose-1-H3 (3.5 mc/smole) was obtained from New England Nuclear Corp., and a-P32-ATP (0.5 mc/14mole) from International Chemical and Nuclear Corp. J