Characterization of DNA strand breakage in vitro by the antitumor protein neocarzinostatin

Abstract

The antitumor protein antibiotic neocarzinostatin causes strand scission of [Micrococcus luteus] DNA in vitro in the presence of a sulfhydryl compound. The breaks are single stranded in nature and bear 5''-phosphoryl termini. All 4 deoxymononucleotides are recoverable at the 5''-ends of the cleavage sites, although a higher proportion of dGMP and TMP are consistently found. The lesions are not repairable with polynucleotide ligase from Escherichia coli. A quantitative assay was developed to determine the pH profile and time course of the reaction. Data from protection experiments with synthetic and natural DNAs indicate the requirement for thymidylic acid and deoxyadenylic acid in the DNA for cutting. In DNA-RNA hybrids, riboadenylic acid can substitute for deoxyadenylic acid, whereas ribouridylic acid cannot substitute for thymidylic acid. Release of thymine is detected, and the amount of release correlates well with the number of strand scissions.