Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

TY - JOUR

T1 - Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

AU - Taschner, Michael

AU - Basquin, Jérôme

AU - Steigenberger, Barbara

AU - Schäfer, Ingmar B.

AU - Soh, Young Min

AU - Basquin, Claire

AU - Lorentzen, Esben

AU - Räschle, Markus

AU - Scheltema, Richard A.

AU - Gruber, Stephan

N1 - Publisher Copyright: © 2021 The Authors. Published under the terms of the CC BY 4.0 license

PY - 2021/8

Y1 - 2021/8

N2 - 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.

AB - 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.

KW - chromosome segregation

KW - cohesion

KW - condensin

KW - loop extrusion

KW - Smc5/6

UR - http://www.scopus.com/inward/record.url?scp=85108879789&partnerID=8YFLogxK

U2 - 10.15252/embj.2021107807

DO - 10.15252/embj.2021107807

M3 - Journal article

C2 - 34191293

AN - SCOPUS:85108879789

SN - 0261-4189

VL - 40

JO - EMBO Journal

JF - EMBO Journal

IS - 15

M1 - e107807

ER -