S 1 nuclease converts relaxed circular closed duplex DNA to open linear DNA of unit length

Superhelical DNA can be cleaved open by the single-strand-specific Sr nuclease from Aspergillus oryzae (EC 3.1.4.X) to form linear duplexes of unit length [l-4] . The suggestion was made, that, owing to a topological constraint in these molecules partially single-stranded structures might be revealed as sites of attack for the Sr nuclease [l-3] . Introduction of one or more single-strand nicks into a superhelical molecule will cause relaxation of the circular DNA and, concordingly, removal of putative single-stranded regions. Such relaxed molecules should no longer be converted to linear duplexes of unit length (form III) by Sr nuclease if the above-mentioned hypothesis were correct. We have shown, however, that nicked circular SV40 DNA (form II) can, nevertheless, be cleaved open to form III by the enzyme, despite the absence of partially weakly base-paired regions and regardless of the number of single-strand nicks present [5]. To test further the hypothesis that S, nuclease requires single-stranded regions in order to introduce a double-strand break into circular DNA, we have employed synthetic circular fd replicative intermediate (RF) DNA [6] as a substrate. This double-stranded DNA is relaxed, is devoid of nicks and of topological constraints, and should, therefore, provide an interesting substrate for the double-strand cleaving activity in Sr nuclease preparations. We will show, that in disagreement with the current hypothesis, Sr nuclease does not reqilire single-stranded regions in order to exert its double-strand-cleaving activity on circular DNA, since fd RF DNA is converted to form III by treatment with Sr nuclease.