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Infection of Escherichia coli by bacteriophage T4 induces a series of new enzymes which are not detectable in the uninfected cell. One such enzyme, polynucleotide ligase, was described in a previous communication.' This enzyme catalyzes the repair of single-strand breaks2 in a DNA duplex by the formation of phosphodiester bonds; in the reaction adenosine 5'-triphosphate (ATP) is cleaved to adenosine 5'-phosphate (AMP) and inorganic pyrophosphate (PPi). An enzyme present in uninfected E. coli cells, which, like the T4 ligase, I 3 catalyzes the covalent joining of polynucleotides, has been reported by Gellert,4 Becker et al.,3 and Olivera and Lehman.5 However, the joining enzyme purified from uninfected cells requires NAD+, instead of ATP, as a cofactor.5 6 After infection of E. coli by phage T4, there is an extensive fragmentation and dispersion of phage DNA among the parental and progeny molecules.7-9 An enzyme such as polynucleotide ligase might be involved in the covalent joining of these recombined fragments. One approach to determining the role of polynucleotide ligase in vivo is to obtain mutants of phage T4 defective in the synthesis of this enzyme and to study DNA metabolism in cells infected with them. If the ligase is important in the metabolism of the phage DNA, it may be essential for the development of the mature phage. Conditional lethal mutants of phage T410 defective in DNA synthesis provide a means for identifying the structural gene for this type of enzyme. Studies with such mutants have already led to the identification of the genes for several enzymes: deoxynucleotide kinase (gene 1),11, 12 deoxycytidylate hydroxymethylase (gene 42),1'3 14 DNA polymerase (gene 43),151 16 and deoxycytidine triphosphatase (gene 56).11 The present report describes the identification of gene 30 as the structural gene for T4 phage-induced polynucleotide ligase. Dr. R. S. Edgar provided us with a number of conditional lethal mutants of bacteriophage T4 which we have examined for their ability to induce polynucleotide ligase. These studies have resulted in the identification of two amber mutants which fail to induce an active ligase in the nonpermissive host, and three temperature-sensitive mutants which induce a ligase more heat-labile than that induced by the wild-type phage. Experimental Procedure.-Materials: Unlabeled and radioactively labeled nucleotides were obtained as previously described.'7 P12-labeled PPi was purchased from New England Nuclear Corp. 5'-Hydroxyl-terminated DNA (substrate for polynucleotide kinase)"8 and T7 DNA containing internal P"2-phosphomonoesters (substrate for polynucleotide ligase)l were prepared as described previously. Methods: (a) Growth of bacteriophages: T4 am H39X was provided by Dr. J. Hosoda; T4 agt was provided by Dr. S. E. Luria. Other bacteriophages and E. coli strains were provided by Dr. R. S. Edgar. Stocks of T4 amber mutants were prepared by infecting E. coli CR63 (the permissive host) in H-medium" at a multiplicity of 0.1. The cultures were grown at 370 until lysis occurred after 5 hr. The lysates were chilled to 40 and cleared of debris by centrifugation for 10 min at 10,000 X

Enzymatic breakage and joining of deoxyribonucleic acid. II. The structural gene for polynucleotide ligase in bacteriophage T4.