26] Exonuclease III of Escherichia coli K-12, an AP endonuclease

Abstract

This chapter reviews exonuclease III of Escherichia coli K-12, which is an AP endonuclease. Exonuclease III of E. coli catalyzes the hydrolysis of several types of phosphoester bonds in duplex DNA. It is a 3' → 5' exonuclease, releasing 5'-mononucleotides from the 3' ends of DNA strands; it is a DNA 3'-phosphatase, hydrolyzing 3'-terminal phosphomonoesters; and it is an AP endonuclease, cleaving phosphodiester bonds at apurinic or apyrimidinic sites to produce new 5' termini that are base-free deoxyribose 5-phosphate residues. In addition, the enzyme has an RNase H activity; it will preferentially degrade the RNA strand in a DNA-RNA hybrid duplex, presumably exonucleolytically. The exonuclease assay is the simplest. It measures the release of acid-soluble radioactive material (mononucleotides) from bacteriophage T7 [ 3 H]DNA. In order to saturate the enzyme and to conserve radiolabeled substrate, the T7 DNA is diluted with unlabeled salmon sperm DNA, and the mixture is sheared by sonication, thereby increasing the concentration of T-hydroxyl termini. The chapter further presents the preparation of substrate.