Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo
- University of Sussex, United Kingdom
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 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.
Data availability
Single molecule traces exported from GDSC SMLM plugin and used for analysis in SpotOn software are available via the Open Science Framework (osf.io/myxtr).
Article and author information
Author details
Thomas J Etheridge
Eduard Campillo-Funollet
Alex David Herbert
Funding
Wellcome Trust (110047/Z/15/Z)
- Antony M Carr
Medical Research Council (MR/P018955/1)
- Antony W Oliver
- Johanne M Murray
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Etheridge et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo
eLife 10:e68579.
https://doi.org/10.7554/eLife.68579
Further reading
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The R-loop is a common transcriptional by-product that consists of an RNA-DNA duplex joined to a displaced strand of genomic DNA. While the effects of R-loops on health and disease are well established, there is still an incomplete understanding of the cellular processes responsible for their removal from eukaryotic genomes. Here, we show that a core regulator of chromosome architecture —the Smc5/6 complex— plays a crucial role in the removal of R-loop structures formed during gene transcription. Consistent with this, budding yeast mutants defective in the Smc5/6 complex and enzymes involved in R-loop resolution show strong synthetic interactions and accumulate high levels of RNA-DNA hybrid structures in their chromosomes. Importantly, we demonstrate that the Smc5/6 complex acts on specific types of RNA-DNA hybrid structures in vivo and promotes R-loop degradation by the RNase H2 enzyme in vitro. Collectively, our results reveal a crucial role for the Smc5/6 complex in the removal of toxic R-loops formed at highly transcribed genes and telomeres.
