Direct observation of strand passage by dna-topoisomerase and its limited processivity

Katsunori Yogo, Taisaku Ogawa, Masahito Hayashi, Yoshie Harada, Takayuki Nishizaka, Kazuhiko Kinosita

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    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.

    Original languageEnglish
    Article numbere34920
    JournalPLoS One
    Volume7
    Issue number4
    DOIs
    Publication statusPublished - 2012 Apr 9

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    ASJC Scopus subject areas

    • Agricultural and Biological Sciences(all)
    • Biochemistry, Genetics and Molecular Biology(all)
    • Medicine(all)

    Cite this

    Yogo, K., Ogawa, T., Hayashi, M., Harada, Y., Nishizaka, T., & Kinosita, K. (2012). Direct observation of strand passage by dna-topoisomerase and its limited processivity. PLoS One, 7(4), [e34920]. https://doi.org/10.1371/journal.pone.0034920