biostudies-literatureHallett STMedical Research Council4534-4549https://www.ebi.ac.uk/biostudies/studies/S-EPMC8096239biostudies-literatureUnknown49(8)The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein 'holo-complex' is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPaseNucleic acids researchNse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex.PMC8096239MR/P018955/1G1001668Schellenberger PZhou LBeuron FHallett STOliver AWMorris EMurray JMfalseNse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex.The multi-component Smc5/6 complex plays a critical role in the resolution of recombination intermediates formed during mitosis and meiosis, and in the cellular response to replication stress. Using recombinant proteins, we have reconstituted a series of defined Saccharomyces cerevisiae Smc5/6 complexes, visualised them by negative stain electron microscopy, and tested their ability to function as an ATPase. We find that only the six protein 'holo-complex' is capable of turning over ATP and that its activity is significantly increased by the addition of double-stranded DNA to reaction mixes. Furthermore, stimulation is wholly dependent on functional ATP-binding pockets in both Smc5 and Smc6. Importantly, we demonstrate that budding yeast Nse5/6 acts as a negative regulator of Smc5/6 ATPase2021-01-01T00:00:00Z2021 May2026-04-18T01:38:06.069Z2022-02-10T18:53:15.029ZS-EPMC80962393384907210.1093/nar/gkab234