title: Direct observation of strand passage by DNA-topoisomerase and its limited processivity.
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 IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51 ± 0.33 µm (11 ± 6 turns) of a braid was unlinked in a burst of reactions taking 8 ± 4 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.
authors.affiliation: Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo, Japan.
authors.forename: Katsunori
authors.initials: K
authors.lastname: Yogo
authors.forename: Taisaku
authors.initials: T
authors.lastname: Ogawa
authors.forename: Masahito
authors.initials: M
authors.lastname: Hayashi
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Harada
authors.forename: Takayuki
authors.initials: T
authors.lastname: Nishizaka
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Kinosita
mesh_terms: D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans
chemical_list: D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D004247:DNA; D004250:DNA Topoisomerases, Type II