[29] Purification and Properties of Escherichia Coli Endodeoxyribonuclease V

This chapter discusses the purification and properties of Escherichia coli endodeoxyribonuclease V. This assay measures the conversion of phage fd single stranded, circular DNA to a linear form which is hydrolyzed to acid soluble products by Escherichia coli exonuclease I. An appropriate amount of endonuclease is added, and then the reaction mixture is incubated for 30 rains at 37°C. It is then chilled on ice, and 0.05 ml of bovine serum albumin (10 mg/ml) and 0.25 ml of 7% TCA are added with mixing following each addition. In addition, the number of endonucleolytic cleavages can be estimated by determining the number of fd DNA molecules opened during the incubation. An fd DNA molecule contains 6389 nucleotides, so the acid-solubilization of 1 nmole of DNA is taken to be due to the conversion of 0.157 pmole of DNA molecules from the circular to the linear form. A minimum number of such cleavages is estimated unless one utilizes a Poisson correction because only one endonucleolytic cleavage is required for each such conversion.