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Nucleic Acids Res. 2014 Jun;42(11):7421-8. doi: 10.1093/nar/gku320. Epub 2014 May 3.

Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure.

Author information

1
Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
2
CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan.
3
Centro Nacional de Biotecnología, CNB-CSIC, C/Darwin 3, 28049 Madrid, Spain.
4
Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan.
5
Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan takeyasu@lif.kyoto-u.ac.jp.

Abstract

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.

PMID:
24792171
PMCID:
PMC4066755
DOI:
10.1093/nar/gku320
[Indexed for MEDLINE]
Free PMC Article

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