Enzymatic Breakage and Joining of Deoxyribonucleic Acid

Abstract

Radioactive labeling of the 5′-termini of DNA with polynucleotide kinase has been used in conjunction with treatment by alkaline phosphatase from Escherichia coli to study single strand breaks (phosphodiester bond interruptions) produced in duplex DNA molecules by the action of pancreatic deoxyribonuclease. At 37° alkaline phosphatase will quantitatively hydrolyze external phosphomonoesters (those located at either end of the duplex molecule), but it will not hydrolyze internal phosphomonoesters (those located at single strand breaks). However, at elevated temperatures or following denaturation of the DNA, both external and internal phosphomonoesters are hydrolyzed by phosphatase. These methods permit (a) the identification, measurement, and characterization of single strand breaks in DNA molecules and (b) the preparation of specifically labeled DNAs used for studies on enzymes acting at internal and external termini.