Study on the reaction mode of restriction endonuclease Eco RI

Abstract

Eco RI, mostly investigated Type II restriction endonuclease, is a phosphodiesterase that hydrolyses the phosphodiester bond of DNA. But, very little is known about the chemical steps involved on the cleavage of the scissible phosphodiester bond. In this research, with both the plasmid DNA and $\lambda$ bacteriophage DNA as substrates, specific reaction mode of Eco RI as a phosphodiesterase for double stranded DNA was surveyed. Enzyme activity was chiefly traced either by film scanning technique followed by the separation of product DNA fragments in agarose gel electrophoresis, or accompanied by newly developed assay method using the HPLC(High Performance Liquid Chromatography) system employed gel permeation column. Simplicity, high sensitivity good reproducibility, and safety are the peculiar advantages of this assay system. It was found that the cleavage of both strands of the duplex DNA was achieved only after two independent phosphodiesterase actions for single strands, which were thought as the same reaction for both the first and the second cleavage. Say, closed circular form DNA was changed into linear form DNA through the intermediate open circular form DNA, and the intermediate form was more accumulated when the condition was more deviated from the optimal state: It was thought that the intermediate was dissociated from the enzyme and this was the transient product of single turn-over of the enzyme. A proposed model mechanism of the above phosphodiesterase action for double stranded DNA by Eco RI restriction endonuclease was suggested from the study of chemical modification and pH-activity relationship through the identification of ionizable groups at the active site of the enzyme. In the chemical modification study, enzyme inactivation by Iodoacetamide and Diethyl pyrocarbonate, and by Diisopropyl fluorophosphate suggested that Histidine and Serine residue of the enzyme were directly involved in the prototropic groups at the active site of Eco RI endo...