Phosphoryl Termini Research Articles

On the mechanism of the polynucleotide joining reaction.

Polynucleotide-joining enzymes (ligases) have been identified in uninfected and phage-infected Escherichia coli (Gellert, 1967; Olivera and Lehman, 1967a; Zimmerman et al., 1967; Gefter et al., 1967; Weiss and Richardson, 1967a; Cozzarelli et al., 1967). Both types of enzyme catalyze the synthesis of a phosphodiester bond between the 3′ hydroxyl and 5′ phosphoryl termini of DNA chains which have been properly aligned in a double-helical structure. Such enzymes are of interest because of their possible involvement in genetic recombination, ‘dark’ repair of ultraviolet-induced lesions, and in DNA synthesis. It has already been demonstrated that the polynucleotide ligase induced by phage T4 is essential for normal T4 DNA replication (Fareed and Richardson, 1967).

The Enzymatic Cleavage of Phosphate Termini from Polynucleotides

1. After infection of Escherichia coli B with T-even bacteriophages, a novel 3'-deoxynucleotidase activity appears. The induction is blocked by the presence of chloramphenicol and is not observed after infection with T-odd bacteriophages. This enzyme has been purified approximately 2000-fold from extracts of E. coli B infected with T4 am 82. In addition to its attack on 3'-deoxymononucleotides, the enzyme also catalyzes the selective removal of 3'-phosphoryl groups from DNA. The enzyme preparations are without effect on 3'-ribomononucleotides, 3'-phosphoryl groups of RNA, or any 5'-phosphate esters. 2. A survey of various strains of E. coli revealed strain C to be a rich source of 3'-nucleotidase activity. This activity was purified approximately 1300-fold and the final preparation was found to possess the following specificity. The enzymatic activity is nonspecific at the mononucleotide level, catalyzing the cleavage of 2',3'- and 5'-substituted ribomononucleotides as well as 3'- or 5'-deoxymononucleotides. The enzyme also cleaves 3',5'-nucleoside diphosphates. In its attack on pTp, the enzyme appears to remove first the 3'-phosphate group before cleaving the 5'-phosphoester bond. This apparent preference for the cleavage of the 3'-phosphate group in the presence of a 5'-phosphate substitution is supported by observations on the specificity of the enzyme with the use of polynucleotides as substrates. Thus, with polynucleotides, only the 2'- or 3'-terminal phosphate groups are removed, while the 5'-phosphoryl termini are not attacked at all. Thus, the purified E. coli C acid phosphatase can be used to measure specifically the 2'- or 3'-phosphate ends of RNA and DNA. 3. After infection of E. coli B with T4 am 82, an enzyme activity which attacks the 5'-phosphate group of RNA and DNA appears. The increase of activity upon infection is prevented if the infection process is carried out in the presence of chloramphenicol. This activity has been purified approximately 3000-fold. The final preparation shows a high degree of specificity in the cleavage of 5'-phosphoryl groups from polynucleotides and is without effect on 5'-mononucleotides. The products formed after the action of the 5'-polynucleotide phosphatase have been identified as inorganic phosphate and 5'-hydroxyl-terminated polynucleotides. The latter were identified by their ability to be phosphorylated in the 5'-hydroxylpolynucleotide kinase reaction. The final preparation of 5'-polynucleotide phosphatase is contaminated with 3'-deoxynucleotidase.