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Single-molecule live cell imaging of the Smc5/6 DNA repair complex

View ORCID ProfileThomas J. Etheridge, Desiree Villahermosa, Anja Irmisch, Adam T. Watson, Alex Herbert, Hung Q. Dang, Mark A. Osborne, Antony W. Oliver, Antony M. Carr, Johanne M. Murray
doi: https://doi.org/10.1101/2020.06.19.148106
Thomas J. Etheridge
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, UK
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  • ORCID record for Thomas J. Etheridge
  • For correspondence: j.m.murray@sussex.ac.uk t.etheridge@sussex.ac.uk
Desiree Villahermosa
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, UK
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Anja Irmisch
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, UK
3Department of Dermatology, University Hospital Zürich, Switzerland
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Adam T. Watson
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, UK
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Alex Herbert
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, UK
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Hung Q. Dang
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, UK
4Texas A&M University, Texas, United States
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Mark A. Osborne
2Chemistry Department, School of Life Sciences, University of Sussex, Falmer, UK
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Antony W. Oliver
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, UK
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Antony M. Carr
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, UK
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Johanne M. Murray
1Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, UK
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  • For correspondence: j.m.murray@sussex.ac.uk t.etheridge@sussex.ac.uk
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Abstract

The Smc5/6 complex is involved in various DNA transactions and is best known for ensuring the fidelity of homologous recombination. We exploit single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association. We show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits we show that the Nse3 double-stranded DNA binding activity and the two arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the ssDNA binding activity at the hinge region does not prevent chromatin association. However, unlike a mutant attenuating chromatin association, a mutant that disrupts ssDNA binding results in highly elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 20, 2020.
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Single-molecule live cell imaging of the Smc5/6 DNA repair complex
Thomas J. Etheridge, Desiree Villahermosa, Anja Irmisch, Adam T. Watson, Alex Herbert, Hung Q. Dang, Mark A. Osborne, Antony W. Oliver, Antony M. Carr, Johanne M. Murray
bioRxiv 2020.06.19.148106; doi: https://doi.org/10.1101/2020.06.19.148106
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Single-molecule live cell imaging of the Smc5/6 DNA repair complex
Thomas J. Etheridge, Desiree Villahermosa, Anja Irmisch, Adam T. Watson, Alex Herbert, Hung Q. Dang, Mark A. Osborne, Antony W. Oliver, Antony M. Carr, Johanne M. Murray
bioRxiv 2020.06.19.148106; doi: https://doi.org/10.1101/2020.06.19.148106

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