Enzymatic Removal and Replacement of Nucleotides at Single Strand Breaks in Deoxyribonucleic Acid

Abstract Pancreatic DNase has been used to introduce single phosphodiester bond interruptions (nicks) into double stranded circular DNA. Such DNA substrates have been used to study the action of several exonucleases at the site of nicks. Escherichia coli exonuclease III and the exonuclease activities of phage T4 DNA polymerase (3' → 5'-nuclease) and E. coli DNA polymerase (3' → 5'- and 5' → 3'-nuclease) initiate hydrolysis at these sites as well as at the termini of linear molecules. Under conditions of limited hydrolysis, these enzymes initiate hydrolysis at all nicks in the molecules. In contrast, phage λ exonuclease is unable to hydrolyze DNA containing nicks. Either the phage T4 or the E. coli DNA polymerase, in the presence of the four deoxyribonucleoside triphosphates, can completely fill in the gaps created by exonuclease action. The combined 5' → 3'-hydrolytic activity and the polymerizing activity of the E. coli DNA polymerase can account for the conversion of a nick displaying a 5'-hydroxyl end group to one displaying a 5'-phosphomonoester; i.e. the nick is translated. These studies have been facilitated by the use of polynucleotide ligase to distinguish nicks containing 5'-phosphomonoesters from gaps and from nicks containing 5'-hydroxyl groups. E. coli alkaline phosphatase does not readily distinguish 5'-phosphomonoesters at gaps from those at nicks; quantitative hydrolysis of phosphomonoesters at these sites by the phosphatase requires a higher temperature than that required for the hydrolysis of phosphomonoesters at the ends of linear duplexes.