Diphosphopyridine nucleotide: a cofactor for the polynucleotide-joining enzyme from Escherichia coli.

The search for enzymes responsible for the joining of DNA molecules has led to the identification and partial purification of an enzyme from Escherichia coli which links preformed polynucleotides through phosphodiester bonds.1 The reaction requires that the polynucleotides to be joined be part of a double-stranded structure, and that a divalent cation and a "cofactor" be added The cofactor, present in extracts of E. coli, was found to be heat-stable, adsorbable to Norit, and could not be replaced by ATP or any other known nucleoside triphosphate. We have now purified the cofactor extensively and found it to be identical with diphosphopyridine nucleotide (DPN). The present report provides evidence for this identification. Materials and Methods.-The preparation of substrates as well as the assay and purification of the polynucleotide-joining enzyme have been described in the previous paper.' Assay of cofactor activity was performed using a reaction mixture identical to that used for assay of the polynucleotide-joining enzyme except that an excess of enzyme (0.1 unit) and a level of cofactor producing approximately half maximal velocity were added.2 One unit of cofactor activity is the amount which promotes the transformation of 1 4;imole of p32 5'-phosphoryl terminus of polydeoxythymidylate (dT) to a form insusceptible to bacterial alkaline phosphatase in 1 minute under these conditions. DPN, reduced DPN (DPNH), triphosphopyridine nucleotide (TPN) (>95%O pure by enzymatic assay), and rabbit muscle lactic dehydrogenase3 were obtained from the Sigma Chemical Co. DPN analogues (3-pyridine aldehyde-, thionicotinamide-, deamino-, and 3-acetyl pyridine deamino-DPN4) were purchased from Mann Research Laboratories. Diadenosine 5'-5' diphosphate (AppA) was the generous gift of Dr. John Moffatt of the Syntex Institute of Molecular Biology. Venom phosphodiesterase5 and N. crassa DPNase6 were purchased from Worthington Biochemical Co. Potato nucleotide pyrophosphatase was purified according to Kornbe-g and Pricer.7 Diethylaminoethyl Sephadex (DEAE-Sephadex) anld Sephadex G-10 were obtained from Pharmacia. Ultraviolet spectra were measured with a Cary recording spectrophotometer, model 14. Radioactive samples were counted using a Nuclear-Chicago model 186 gas-flow counter equipped with a micromil window. Results and Discussion.-Purification of cofactor: An extract (70 ml) of E. coli (strain 1100, generously provided by Professor H. Hoffman-Berling) was prepared as described previously' except that the cells were suspended in 0.01 M Tris-HCl buffer pH 8.0 containing 0.005M ,B-mercaptoethanol and 0.001 M ethylenediaminetetraacetate (EDTA). The extract was brought to pH 4.5 by the addition of 1 N HC1 and the precipitated material was removed by centrifugation. The super-