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Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo

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  • Thomas J Etheridge
  • Desiree Villahermosa
  • Eduard Campillo-Funollet
  • Alex David Herbert
  • Anja Irmisch
  • Adam T Watson
  • Hung Q Dang
  • Mark A Osborne
  • Antony W Oliver
  • Antony M Carr
  • Johanne M Murray
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Article numbere68579
<mark>Journal publication date</mark>16/04/2021
<mark>Journal</mark>eLife
Volume10
Number of pages19
Publication StatusPublished
<mark>Original language</mark>English

Abstract

The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using 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 arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.