Direct Observation of Strand Passage by DNA- Topoisomerase and Its Limited Processivity

Abstract

Abstract Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIa, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.5160.33 mm (1166 turns) of a braid was unlinked in a burst of reactions taking 864 s, the unlinked length being essentially independent of the enzyme concentration between 0.25-37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ,100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ,10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ,10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments