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

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 unchall...

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Published in:eLife Vol. 10
Main Authors: Etheridge, Thomas J, Villahermosa, Desiree, Campillo-Funollet, Eduard, Herbert, Alex David, Irmisch, Anja, Watson, Adam T, Dang, Hung Q, Osborne, Mark A, Oliver, Antony W, Carr, Antony M, Murray, Johanne M
Format: Journal Article
Language:English
Published: England eLife Science Publications, Ltd 16.04.2021
eLife Sciences Publications Ltd
eLife Sciences Publications, Ltd
Subjects:
Adenosine triphosphatase
Cell Cycle Proteins - metabolism
Chromatin
Chromatin - metabolism
Chromosome rearrangements
Chromosomes and Gene Expression
DNA damage
DNA repair
Experiments
Genetics and Genomics
Homologous recombination
Lasers
Localization
Microscopy
Proteins
Replication
Schizosaccharomyces - metabolism
Schizosaccharomyces pombe Proteins - metabolism
Single Molecule Imaging
single-molecule microscopy
Single-stranded DNA
SMC proteins
Yeast
genetics
S. pombe
chromosomes
SMC proteins
genomics
single-molecule microscopy
DNA repair
gene expression
ISSN:2050-084X, 2050-084X
Online Access:Get full text
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Summary: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.
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Department of Dermatology, University Hospital Zürich, Switzerland.
ISSN:2050-084X
2050-084X
DOI:10.7554/eLife.68579