Single Molecule Measurements of DNA Decatenation by the Topoisomerase III-RecQ Helicase Complex

Abstract

Topoisomerase III (Topo III) is an ATP-independent enzyme that cleaves a single strand of duplex DNA to relieve torsional strain. Topo III is able to unlink DNA catenanes if one strand contains a single stranded region, but not if both strands are intact. Topo III, however, can decatenate intact DNA when RecQ helicase is present. In vivo this complex is involved in resolution of late replication intermediate linkages and double Holliday junctions. More generally, the unique functions of the Topo III-RecQ complex have been shown to be important for preserving genome stability.We directly measured the effect of RecQ on Topo III's decatenase activity using a magnetic tweezer assay in which two strands of DNA attached to a single bead are interwound to produce a DNA braid. These DNA braids topologically mimic catenated DNA and thus provide an ideal substrate for measuring unlinking at the single molecule level. We tested the effect of RecQ on the rate of Topo III decatenation and unwinding activity. In addition we measured decatenation of intact DNA by the RecQ-Topo III complex. We compared these results to the effect of RecQ on two other topoisomerases that decatenate DNA but are not known to interact with RecQ: topoisomerase I, which acts on DNA with a single stranded region, and topoisomerase IV, which acts on intact DNA. We also probed the effect of crossover geometry on the decatenation activity of the Topo III-RecQ complex. Our results provide insight into the functional interactions between Topo III and RecQ.