Generation of Double-stranded Breaks in Hypernegatively Supercoiled DNA by Drosophila Topoisomerase IIIβ, a Type IA Enzyme

Abstract

Drosophila topoisomerase (topo) III␤ 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 III␤ 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 III␤ 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 III␤ cleavage activity is concentrated primarily in highly A/T-rich regions. These results suggest that topo III␤ may function as a reversible endonuclease in vivo by recognizing and cleaving/rejoining DNA structures with single-stranded character.Processes such as replication, transcription, chromosome segregation, and recombination require the activities of a class of enzymes called topoisomerases (topos).1 Topoisomerases are classified as either type I or type II enzymes, depending on overall structure and mechanism (reviewed in Refs. 1-3). These enzymes work by going through two cycles of transesterification. The first one is initiated by the active-site tyrosine to form a phosphodiester bond with the 5Ј-or 3Ј-DNA phosphate at the transient break site, and the second one is the reversal in which the 3Ј-or 5Ј-hydroxyl at the break reforms the DNA backbone bond, thus regenerating the free tyrosine in the enzyme. The type I enzymes are usually monomers that work by making a reversible single-stranded break in the DNA backbone, whereas type II enzymes are dimeric in structure and thus can introduce a transient double-stranded break. Passage of an intact strand(s) through the broken strand(s) followed by religation of the broken ends completes the topoisomerase reaction cycle and affects the topological transformation in DNA. The type I enzymes are further divided into the IA and IB families, with the Escherichia coli protein as the prototypical type IA enzyme (4, 5). The first eukaryotic type IA topoisomerase was discovered in yeast through a genetic screen for suppression of recombination among repeated sequences (6), and more type IA enzymes (the topo III enzymes) subsequently have been identified in other higher eukaryotes (7-11). Prokaryotes like E. coli and lower eukaryotes such as yeast each possess one topo III enzyme, whereas the metazoans like worms, fruit flies, mice, and humans contain two topo III isoforms, designated topo III␣ and topo III␤.Although much is known about topo I a...

RecQ Helicase Stimulates Both DNA Catenation and Changes in DNA Topology by Topoisomerase III

“…A second single-strand passage event with the opposite unwound strand achieves full catenation of the second molecule. Interestingly, Drosophila topoisomerase III␤ has recently been shown to produce reversible double-stranded DNA breaks (51), which is the type of activity needed to achieve a catenation reaction comparable with that we observe for the E. coli proteins.…”

TDP2/TTRAP Is the Major 5′-Tyrosyl DNA Phosphodiesterase Activity in Vertebrate Cells and Is Critical for Cellular Resistance to Topoisomerase II-induced DNA Damage

“…Similarly, the activity of TDP2 on 5′-phosphotyrosyl SSBs may be physiologically relevant because Top2 also induces this type of break, due most likely to incomplete temporal coordination in the cleavage activity of the two Top2 subunits that comprise the active homodimer (4). In addition, topoisomerase III also creates DNA breaks with 5′-phosphotyrosyl termini and preferentially does so in single stranded DNA (15, 16). …”

Characterization of a unique type IA topoisomerase in Bacillus cereus

“…The relaxation product formed by this enzyme never exceeds the superhelical density of naturally occurring plasmid substrates. Interestingly, dr TOP3β is also able to cleave R‐loops and D‐loops and to generate double‐stranded DNA breaks in hypernegatively supercoiled DNA (Wilson‐Sali and Hsieh, 2002a,b). The depletion of dr TOP3β in Drosophila had no effect on viability or fertility (Wilson et al ., 2000).…”