Functional domains required for the Saccharomyces cerevisiae Mus81-Mms4 endonuclease complex formation and nuclear localization
Functional domains required for the Saccharomyces cerevisiae Mus81-Mms4 endonuclease complex formation and nuclear localization
复制标题
DOI：
10.1016/j.dnarep.2003.08.013
复制
发表时间：
2003-12-09
期刊：
影响因子：
3.8
通讯作者：
Xiao, W
中科院分区：
文献类型：
--
作者：
Fu, Y;Xiao, W
酿酒酵母菌Mms4和Mus81蛋白形成特异性复合物，作为支链DNA分子特异性的核酸内切酶，保护细胞免受DNA烷基化损伤的杀伤，但对电离辐射引起的损伤不起作用。为了进一步了解Mus81-Mms4复合体的结构和功能，我们试图通过缺失和突变分析来定义复合体形成和核定位所需的结构域。酵母双杂交和共免疫沉淀实验表明，Mus81和Mms4的c端100个氨基酸对于异源二聚体的形成都是必需的和足够的。然而，Mms4在n端区域的单个氨基酸取代能够消除相互作用，这表明三维结构对于Mms4与Mus81相互作用也很重要。通过与绿色荧光蛋白的融合和体内亚细胞定位研究，我们证明了Mms4和Mus81是核蛋白，可以独立定位到细胞核中。缺失分析表明，Mms4中两个假定的核定位信号（残基244-263）中的一个是定位所必需的，而Mus81的n端一半是其定位到细胞核所必需的和充分的。(C) 2003 Elsevier B.V.版权所有
The Saccharomyces cerevisiae Mms4 and Mus81 proteins form a specific complex, which functions as an endonuclease specific for branched DNA molecules and protects cells from killing by DNA alkylation damage, but not damage induced by ionizing radiations. In an effort to further understand the structure and functions of the Mus81-Mms4 complex, we attempted to define domains required for complex formation and nuclear localization through deletion and mutagenesis analyses. Combined yeast two-hybrid and co-immunoprecipitation experiments indicate that the C-terminal 100 amino acids of both Mus81 and Mms4 are required and sufficient for heterodimer formation. However, a single amino acid substitution in Mms4 in the N-terminal region is able to abolish the interaction, which suggests that the three-dimensional structure is also important for Mms4 to interact with Mus81. By fusion to green fluorescent protein and in vivo subcellular localization studies, we demonstrate that Mms4 and Mus81 are nuclear proteins and can be localized to the nucleus independently. Deletion analyses indicate that one of two putative nuclear localization signals (residues 244-263) in Mms4 is required for localization, whereas the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus. (C) 2003 Elsevier B.V. All rights reserved.