USE OF UV SPECTROSCOPY FOR THE STUDY OF NUCLEIC-ACID CLEAVAGE BY ESCHERICHIA-COLI RNASE-H AND RESTRICTION ENDONUCLEASES

A one-step spectrophotometric method for monitoring of nucleic acid cl eavage by ribonuclease H from E.coli and type II restriction endonucle ases has been proposed. It is based on recording of the increase in th e UV absorbance at 260 nm during the course of enzymatic reaction. Dup lexes stable under the reaction conditions were chosen as substrates f or the enzymes being studied. In order to obtain duplex dissociation f ollowing their cleavage by the enzyme appreciate temperature condition s were selected. The spectrophotometric method may be applied for rapi d testing of the nuclease activity in protein preparations as well as for precise quantitative analysis of nucleic acid degradation by enzym es. This method may be successfully employed in kinetic studies of nuc leic acid - protein interactions.