Center for Frontier Research, National Institute of Genetics, 1111, Yata, Mishima, Shizuoka 411-8540, Japan.
Center for Frontier Research, National Institute of Genetics, 1111, Yata, Mishima, Shizuoka 411-8540, Japan; Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies), 1111, Yata, Mishima, Shizuoka 411-8540, Japan.
Cell Rep. 2020 Nov 10;33(6):108357. doi: 10.1016/j.celrep.2020.108357.
Cohesin, a critical mediator of genome organization including sister chromatid cohesion, is a ring-shaped multi-subunit ATPase that topologically embraces DNA. Its loading and function on chromosomes require the Scc2-Scc4 loader. Using biochemical reconstitution, we show here that the ability of the loader to bind DNA plays a critical role in promoting cohesin loading. Two distinct sites within the Mis4 subunit are found to cooperatively bind DNA. Mis4 initially forms a tertiary complex with cohesin on DNA and promotes subsequent topological DNA entrapment by cohesin through its DNA binding activity, a process that requires an additional DNA binding surface provided by Psm3, the ATPase domain of cohesin. Furthermore, we show that mutations in the two DNA binding sites of Mis4 impair the chromosomal loading of cohesin. These observations demonstrate the physiological importance of DNA binding by the loader and provide mechanistic insights into the process of topological cohesin loading.
黏合蛋白是一种关键的基因组组织介质，包括姐妹染色单体黏合，它是一种环形多亚基 ATP 酶，可以使 DNA 产生拓扑结构。其在染色体上的加载和功能需要 Scc2-Scc4 加载器。在这里，我们使用生化重建的方法表明，加载器结合 DNA 的能力在促进黏合蛋白加载方面起着关键作用。在 Mis4 亚基内发现了两个不同的 DNA 结合位点，它们协同结合 DNA。Mis4 最初与 DNA 上的黏合蛋白形成三级复合物，并通过其 DNA 结合活性促进随后的拓扑 DNA 捕获，这一过程需要由 Psm3（黏合蛋白的 ATP 酶结构域）提供的额外 DNA 结合表面。此外，我们还表明，Mis4 中两个 DNA 结合位点的突变会损害黏合蛋白在染色体上的加载。这些观察结果表明了加载器的 DNA 结合的生理重要性，并为拓扑黏合蛋白加载的过程提供了机制上的见解。
