Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection.

Taschner, M.; Basquin, J.; Steigenberger, B.; Schaefer, I.; Soh, Y.-M.; Basquin, C.; Lorentzen, E.; Raeschle, M.; Scheltema, R. A.; Gruber, S.

doi:10.1101/2021.02.09.430422

bioRxiv · 10.1101/2021.02.09.430422

Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection.

Taschner, M.·Basquin, J.·Steigenberger, B.·Schaefer, I.·Soh, Y.-M.·Basquin, C.·Lorentzen, E.·Raeschle, M.·Scheltema, R. A.·Gruber, S.

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Abstract

Eukaryotic cells employ three SMC 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.

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Taschner, M., Basquin, J., Steigenberger, B., Schaefer, I., Soh, Y.-M., Basquin, C., Lorentzen, E., Raeschle, M., Scheltema, R. A., Gruber, S.. 2021-02-09. Nse5/6 inhibits the Smc5/6 ATPase to facilitate DNA substrate selection.. https://doi.org/10.1101/2021.02.09.430422

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