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

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

eLife 2021 Apr 16

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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. © 2021, Etheridge et al.

Citation

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. Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo. eLife. 2021 Apr 16;10