I Lehman

DNA ligase of E. coli is a polypeptide of molecular weight 75,000. The comparable T4-induced enzyme is somewhat smaller (63,000 to 68,000). Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA, coupled to the cleavage of the pyrophosphate bond of DPN (E. coli) or ATP (T4). Phosphodiester bond synthesis catalyzed by both enzymes occurs in a series of these discrete steps and involves the participation of two covalent intermediates (Fig. 1). A steady state kinetic analysis of the reaction-catalyzed E. coli ligase supports this mechanism, and further demonstrates that enzyme-adenylate and DNA-adenylate are kinetically significant intermediates on the direct path of phosphodiester bond synthesis. A strain of E. coli with a mutation in the structural gene for DNA ligase which results in the synthesis of an abnormally thermolabile enzyme is inviable at 42 degrees C. Although able to grow at 30 degrees C, the mutant is still defective at this temperature in its ability to repair damage to its DNA caused by ultraviolet irradiation and by alkylating agents. At 42 degrees C, all the newly replicated DNA is in the form of short 10S "Okazaki fragments," an indication that the reason for the mutant's failure to survive under these conditions is its inability to sustain the ligation step that is essential for the discontinuous synthesis of the E. coli chromosome. DNA ligase is therefore an essential enzyme required for normal DNA replication and repair in E. coli. Purified DNA ligases have proved to be useful reagents in the construction in vitro of recombinant DNA molecules.

Abstract Double stranded DNA serves as a template primer for Escherichia coli DNA polymerase when the DNA contains a single strand break (a nick) with a 3'-hydroxyl terminus. Initiation of replication entails covalent extension of the 3'-hydroxyl terminus and a concurrent 5' → 3' nuclease action by the enzyme. The primer strand is hydrolyzed at the 5' side of the nick while the synthetic activity catalyzes the addition of nucleotides to the 3' side. The ability of the enzyme to promote hydrolysis and synthesis simultaneously results in the translation of the nick along the DNA duplex in the 5' to 3' direction. The mechanism for conserving the 5'-strand and providing net synthesis of DNA in later phases of the reaction has not yet been clarified.

There are a number of enzymes present in cell-free extracts of Escherichia coli which can degrade deoxyribonucleic acid (DNA) and smaller polynucleotides derived from it (l-3). One of these enzymes, although unable to hydrolyze high molecular-weight DNA at an appreciable rate, will rapidly hydrolyze DNA which has undergone some prior degradation; it is in this respect analogous to the classical phosphodiesterase of snake venom (4). The purpose of this report is to describe in detail the purification and properties of this enzyme which will be referred to as the Escherichia coli phosphodiesterase. This diesterase has been found to hydrolyze E. coli and calf thymus DNA’s to their constituent 5’-mononucleotides once these polymers have undergone some degradation as a result either of heating or limited treatment with pancreatic DNase. The E. coli phosphodiesterase is also capable of degrading appropriately pretreated bacteriophage DNA’s bearing glucosylated hydroxymethyl cytosine to their constituent mononucleotides. In this respect, it differs from the venom diesterase, which is unable to catalyze the cleavage of most of the linkages in which glucosylated hydroxymethyl cytosine is involved (5-7). In further contrast to the venom diesterase, the E. coli phosphodiesterase cannot hydrolyze either free dinucleotides or the 5’-terminal dinucleotide portion of a polydeoxynucleotide chain.