Minamino Masashi, Higashi Torahiko L, Bouchoux Céline, Uhlmann Frank
Chromosome Segregation Laboratory, The Francis Crick Institute, London, UK.
Life Sci Alliance. 2018 Oct 26;1(5). doi: 10.26508/lsa.201800143.
The ring-shaped chromosomal cohesin complex holds sister chromatids together by topological embrace, a prerequisite for accurate chromosome segregation. Cohesin plays additional roles in genome organization, transcriptional regulation and DNA repair. The cohesin ring includes an ABC family ATPase, but the molecular mechanism by which the ATPase contributes to cohesin function is not yet understood. Here we have purified budding yeast cohesin, as well as its Scc2-Scc4 cohesin loader complex, and biochemically reconstituted ATP-dependent topological cohesin loading onto DNA. Our results reproduce previous observations obtained using fission yeast cohesin, thereby establishing conserved aspects of cohesin behavior. Unexpectedly, we find that non-hydrolyzable ATP ground state mimetics ADP·BeF, ADP·BeF and ADP·AlF, but not a hydrolysis transition state analog ADP·VO , support cohesin loading. The energy from nucleotide binding is sufficient to drive the DNA entry reaction into the cohesin ring. ATP hydrolysis, thought to be essential for cohesin loading, must serve a subsequent reaction step. These results provide molecular insight into cohesin function and open new experimental opportunities that the budding yeast model affords.
环形染色体黏连蛋白复合体通过拓扑环绕将姐妹染色单体结合在一起，这是准确进行染色体分离的一个先决条件。黏连蛋白在基因组组织、转录调控和DNA修复中发挥着额外的作用。黏连蛋白环包含一个ABC家族ATP酶，但其ATP酶促进黏连蛋白功能的分子机制尚不清楚。在这里，我们纯化了芽殖酵母黏连蛋白及其Scc2-Scc4黏连蛋白装载复合体，并在生化层面上重建了依赖ATP的拓扑黏连蛋白加载到DNA上的过程。我们的结果重现了之前使用裂殖酵母黏连蛋白获得的观察结果，从而确立了黏连蛋白行为的保守方面。出乎意料的是，我们发现不可水解的ATP基态模拟物ADP·BeF、ADP·BeF和ADP·AlF，而不是水解过渡态类似物ADP·VO，支持黏连蛋白加载。核苷酸结合产生的能量足以驱动DNA进入黏连蛋白环的反应。ATP水解，虽然被认为对黏连蛋白加载至关重要，但必定参与后续的反应步骤。这些结果为黏连蛋白的功能提供了分子层面的见解，并开启了芽殖酵母模型所提供的新的实验机会。
