Interaction between DNA and Escherichia coli DNA topoisomerase I. Formation of complexes between the protein and superhelical and nonsuperhelical duplex DNAs.

At least two types of complexes are detectable between Escherichia coli DNA topoisomerase I (w protein) and double-stranded DNA. If the duplex DNA is highly negatively supercoiled, the enzyme forms a complex with properties similar to its complex with singlestranded DNA: it is stable in a concentrated NaCl or CsCl solution, but addition of a minute amount of Mg(I1) to the salt solution readily dissociates the complex. Upon addition of sodium dodecyl sulfate or alkali to such a complex, a single-chain scission is generated in the DNA, and the enzyme is found linked covalently to the 5’ phosphoryl side of the scission. With alkali, the pH at which this scission occurs is about 11. This complex is termed the “alkali-cleavable” complex. Pronase treatment of the complex initially causes the removal of a large part of the bound enzyme, but the alkali cleavability of the complex is retained. Extensive pronase digestion of the complex leads to cleavage of the DNA chain and the linking of the remnant of the protein to the 5’ side. With nonsuperhelical DNA, the enzyme forms a different type of complex. Although stable in concentrated salt solutions, the second type of complex is neither dissociated by the addition of Mg(I1) nor does DNA chain scission occur when protein denaturants are added. The strong dependence of the formation of the alkali-cleavable complex on negative superhelicity suggests that unwinding of the DNA duplex is necessary for the formation of this complex. It is likely that this complex represents an intermediate in the topoisomerization of DNA by the enzyme.