Enzymatic joining of DNA strands, II. An enzyme-adenylate intermediate in the dpn-dependent DNA ligase reaction.

Abstract

Recent work in 2 and other. laboratories3-6 has demonstrated the existence of enzymes which catalyze the covalent closure of single-strand breaks in duplex DNA. Interest in such enzymes derives in part from their probable biological importance; thus, the enzyme induced by infection with bacteriophage T4 has been shown to be necessary for multiplication of the phage.7 Characterization of purified enzymes from uninfected2' 4 5 and T4-infected3 Escherichia coli has revealed a requirement in each case for a nucleotide cofactor. The enzyme from uninfected cells specifically requires DPN,2 I which is broken down into AMP and NMN in the course of the reaction; the enzyme from T4infected cells, by contrast, splits ATP into AMP and PPi.3 In both cases, 3'-5' plhosphodiester bonds are formed by covalent joining of the two apposed DNA termini at a single-strand break. This class of enzymes thus catalyzes a complex reaction which may be expected to proceed in several distinguishable stages. This report describes the first step of the reaction catalyzed by the DNA ligase of uninfected E. coli. Upon admixture with DPN in the absence of DNA, the enzyme forms a stable enzyme-adenylate complex, releasing NMN. The enzyme-adenylate complex, as isolated by gel filtration, can seal single-strand breaks in DNA in the absence of DPN. AMP is released in this reaction. The enzyme-adenylate complex may also be discharged with NMN to yield DPN by reversal of the reaction which formed the complex. As a consequence an exchange between DPN and NMN can be observed, as noted by Olivera and Lehman.5 These reactions are summarized in the following equations: