Shwartz Michal, Matityahu Avi, Onn Itay
Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, 1311502, Israel.
Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, 1311502, Israel
G3 (Bethesda). 2016 Aug 9;6(8):2655-63. doi: 10.1534/g3.116.031674.
Cohesin is a multi-subunit complex that plays an essential role in genome stability. Initial association of cohesin with chromosomes requires the loader-a heterodimer composed of Scc4 and Scc2 However, very little is known about the loader's mechanism of action. In this study, we performed a genetic screen to identify functional domains in the Scc4 subunit of the loader. We isolated scc4 mutant alleles that, when overexpressed, have a dominant negative effect on cell viability. We defined a small region in the N terminus of Scc4 that is dominant negative when overexpressed, and on which Scc2/Scc4 activity depends. When the mutant alleles are expressed as a single copy, they are recessive and do not support cell viability, cohesion, cohesin loading or Scc4 chromatin binding. In addition, we show that the mutants investigated reduce, but do not eliminate, the interaction of Scc4 with either Scc2 or cohesin. However, we show that Scc4 cannot bind cohesin in the absence of Scc2 Our results provide new insight into the roles of Scc4 in cohesin loading, and contribute to deciphering the loading mechanism.
黏连蛋白是一种多亚基复合体，在基因组稳定性中发挥着至关重要的作用。黏连蛋白与染色体的初始结合需要装载蛋白——一种由Scc4和Scc2组成的异二聚体。然而，关于装载蛋白的作用机制却知之甚少。在本研究中，我们进行了一项遗传筛选，以确定装载蛋白Scc4亚基中的功能结构域。我们分离出了scc4突变等位基因，当这些基因过表达时，会对细胞活力产生显性负效应。我们在Scc4的N端定义了一个小区域，该区域过表达时具有显性负效应，且Scc2/Scc4的活性依赖于此。当突变等位基因以单拷贝形式表达时，它们是隐性的，无法支持细胞活力、黏连、黏连蛋白装载或Scc4与染色质的结合。此外，我们发现所研究的突变体会减少，但不会消除，Scc4与Scc2或黏连蛋白之间的相互作用。然而，我们发现，在没有Scc2的情况下，Scc4无法结合黏连蛋白。我们的研究结果为Scc4在黏连蛋白装载中的作用提供了新的见解，并有助于阐明装载机制。
