Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

尽管在姐妹染色单体内聚力和染色体组织中起关键作用，但内聚素环加载到 DNA 上的机制仍然未知。在这里，我们结合生化方法和低温电子显微镜 (cryo-EM) 来可视化一个内聚素加载中间体，其中 DNA 被锁定在通向内聚素环的两个门之间。在这个结构框架的基础上，我们设计了实验来建立内聚加载过程中的事件顺序。在初始步骤中，DNA 穿过 N 端 kleisin 门，该门首先在 ATP 结合时打开，然后随着内聚蛋白加载器将 DNA 锁定在 ATP 酶门上而关闭。ATP 水解将导致 ATPase 门打开以完成 DNA 进入。DNA 加载是否成功或导致环挤出可能由引导 DNA 通过 kleisin 门的保守 kleisin N 末端尾部决定。我们的结果建立了粘附素加载到 DNA 上的分子基础。