@article{32530563914a477889eb7ab192817e13,
title = "Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding",
abstract = "Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.",
keywords = "Smc5/6, chromosome segregation, cohesion, condensin, loop extrusion",
author = "Michael Taschner and J{\'e}r{\^o}me Basquin and Barbara Steigenberger and Sch{\"a}fer, {Ingmar B} and Young-Min Soh and Claire Basquin and Esben Lorentzen and Markus R{\"a}schle and Scheltema, {Richard A} and Stephan Gruber",
note = "Funding Information: We thank members of the Gruber laboratory for comments on the manuscript, the Protein Analysis Facility (PAF) at FBM‐UNIL for the identification of Nse5 and Nse6 fragments, the crystallization facility at the MPI of Biochemistry for assistance with crystal screening/optimization and the staff at the Swiss Light Source (SLS) for help with data collection. We are grateful to Serge Pelet and the Pelet laboratory for helpful advice and for sharing materials and reagents for genetic engineering in yeast. This work was supported by the Swiss National Science Foundation (310030L_170242), the European Research Council (Horizon 2020 ERC CoG 724482) to S.G and the German Research Foundation (DFG RA2941/1‐1) to M.R. Additional support came from the Netherlands Organisation for Scientific Research (NWO) research programme TA with project number 741.018.201 and the European Union Horizon 2020 programme INFRAIA project Epic‐XS (Project 823839) to R.A.S. Funding Information: We thank members of the Gruber laboratory for comments on the manuscript, the Protein Analysis Facility (PAF) at FBM-UNIL for the identification of Nse5 and Nse6 fragments, the crystallization facility at the MPI of Biochemistry for assistance with crystal screening/optimization and the staff at the Swiss Light Source (SLS) for help with data collection. We are grateful to Serge Pelet and the Pelet laboratory for helpful advice and for sharing materials and reagents for genetic engineering in yeast. This work was supported by the Swiss National Science Foundation (310030L_170242), the European Research Council (Horizon 2020 ERC CoG 724482) to S.G and the German Research Foundation (DFG RA2941/1-1) to M.R. Additional support came from the Netherlands Organisation for Scientific Research (NWO) research programme TA with project number 741.018.201 and the European Union Horizon 2020 programme INFRAIA project Epic-XS (Project 823839) to R.A.S. Publisher Copyright: {\textcopyright} 2021 The Authors. Published under the terms of the CC BY 4.0 license",
year = "2021",
month = aug,
day = "2",
doi = "10.15252/embj.2021107807",
language = "English",
volume = "40",
pages = "1--23",
journal = "EMBO Journal",
issn = "0261-4189",
publisher = "Nature Publishing Group",
number = "15",
}