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

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.

真核细胞依靠三类染色体结构维持（SMC，Structural Maintenance of Chromosomes）复合物来管控DNA折叠与拓扑状态。Smc5/6复合物在DNA修复及抑制有害DNA连接体积累方面发挥功能。为阐明Smc5/6复合物的特定特征如何调控上述功能，我们重构了酵母全复合物。研究发现，Nse5/6亚复合物通过阻断功能性ATP结合，强力抑制Smc5/6的ATP酶（ATPase）活性。该抑制作用可被质粒DNA结合解除，但无法被短链线性DNA解除；而在缺失Nse5/6的条件下，则观测到相反效应。基于Nse5/6的晶体结构与交联质谱（cross-linking mass spectrometry）数据，我们揭示了Smc5/6上存在两个Nse5/6结合位点：一个位于Smc5/6的臂区，另一个位于头部区域，后者大概率对ATP水解产生抑制作用。半胱氨酸交联（cysteine cross-linking）实验证实，与Nse5/6的相互作用将ATP酶结构域锚定在非活性状态，该状态可被ATP与DNA解除稳定。在类似实验条件下，Nse4/3/1模块从ATP酶结构域上脱离。综上，我们阐明了Nse5/6通过直接抑制Smc5/6的ATP酶活性，从而调控DNA底物选择的分子机制。