Generation of double-stranded breaks in hypernegatively supercoiled DNA by Drosophila topoisomerase IIIβ, a type IA enzyme

Abstract

Drosophila topoisomerase (topo) IIIβ is a member of the type IA family of DNA topoisomerases, which generates a single-stranded break to form a covalent complex with the 5′-end of DNA. We show here that a purified preparation of topo IIIβ is able to convert a hypernegatively supercoiled substrate into primarily nicked, but also linear, DNA at enzyme/DNA molar ratios of 5:1 or greater. Although the optimal temperature for the relaxation activity is between 37 and 45 °C, maximal cleavage occurs between 23 and 30 °C, a temperature range that is more physiologically relevant for fruit flies. The cleavage products require protease treatment to enter the gel, they are stable over time, they are reversible, and they are not observed with a Y332F active site mutant, which further supports the idea that topo IIIβ possesses an endonucleolytic cleavage activity. This cleavage activity appears to be specific for highly unwound, or single strand-containing substrates. Southern blot analysis of the cleavage products demonstrates that the topo IIIβ cleavage activity is concentrated primarily in highly A/T-rich regions. These results suggest that topo IIIβ may function as a reversible endonuclease in vivo by recognizing and cleaving/rejoining DNA structures with single-stranded character.