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Junction resolving enzymes use multivalency to keep the Holliday junction dynamic

Nature Chemical Biology volume 15, pages 269–275 (2019)Cite this article

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Abstract

Holliday junction (HJ) resolution by resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single-molecule fluorescence resonance energy transfer analysis of resolving enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.

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Fig. 1: Endo I binding to HJs captures the instantaneous junction conformer and permits exchange between two isoforms (B1 and B2).
Fig. 2: Endo I binding captures the instantaneous branch position and permits branch migration through a PD intermediate.
Fig. 3: RuvC binding permits conformer exchange and branch migration through PD.
Fig. 4: GEN1 and hMus81-Eme1 binding both permit conformer exchange and hMus81-Eme1 binding also permits branch migration.
Fig. 5: Proposed models for the coordination of junction resolution, conformer exchange and branch migration.

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Code availability

All custom software and codes are available from the corresponding authors on request or can be downloaded from the Ha Research Group website at http://ha.med.jhmi.edu/resources/.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We acknowledge Ha lab members for experimental help and discussion. This work was supported by grants from the National Science Foundation (no. PHY-1430124) and the National Institutes of Health (no. GM 122569) to T.H., and grants from the Korean government (no. NRF 2018R1A2A1A190 to Y.C.). R.Z. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. T.H. is an employee of the Howard Hughes Medical Institute. Work in the Lilley lab is funded by Cancer Research UK program grant no. A18604.

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Authors and Affiliations

  1. Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Champaign, IL, USA

    Ruobo Zhou & Taekjip Ha

  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    Ruobo Zhou

  3. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, MD, USA

    Olivia Yang & Taekjip Ha

  4. Cancer Research UK Nucleic Acid Structure Research Group, School of Life Sciences, The University of Dundee, Dundee, UK

    Anne-Cécile Déclais, Alasdair D. J. Freeman & David M. J. Lilley

  5. Department of Life Science, Pohang University of Science and Technology, Pohang, South Korea

    Hyeonseok Jin, Gwang Hyeon Gwon & Yunje Cho

  6. Howard Hughes Medical Institute, Baltimore, MD, USA

    Taekjip Ha

  7. Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA

    Taekjip Ha

  8. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA

    Taekjip Ha

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Contributions

R.Z. and T.H. conceived and designed the study. R.Z. and O.Y. performed experiments and analyzed the data. A.-C.D., A.D.J.F. and D.M.J.L. expressed and purified wild type Endo I proteins, Endo I mutants and GEN1 proteins. A.-C.D. performed Endo I gel retardation assays. H.J., G.H.G. and Y.C. expressed and purified hMus81-Eme1 proteins. R.Z. and T.H. wrote the manuscript with input from the other authors.

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Correspondence to Ruobo Zhou or Taekjip Ha.

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Zhou, R., Yang, O., Déclais, AC. et al. Junction resolving enzymes use multivalency to keep the Holliday junction dynamic. Nat Chem Biol 15, 269–275 (2019). https://doi.org/10.1038/s41589-018-0209-y

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