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Properties of strand breakage in duplex and single-stranded DNA by the wheat germ type 1 DNA topoisomerase were investigated. Strand breakage in duplex DNA is dependent upon the use of denaturing conditions to inactivate the enzyme and terminate the reaction, whereas breakage of single-stranded DNA occurs under the normal reaction conditions and is not dependent upon denaturation. Breakage generates a free 5′ hydroxyl group and enzyme bound to the 3′ side of the break, presumably via the 3′ phosphate group. The location of sites of breakage with both duplex and single-stranded DNA is not random. In all these respects the wheat germ enzyme closely resembles the rat liver type 1 topoisomerase. A comparison of the locations of the sites of breakage in duplex DNA generated by the topoisomerases from wheat germ and rat liver indicates a number of common sites, although the patterns of breakage are not identical.