Requirements for the interaction of mouse polκ with ubiquitin and its biological significance
Requirements for the interaction of mouse polκ with ubiquitin and its biological significance
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DOI：
10.1074/jbc.m709275200
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发表时间：
2008-02-22
影响因子：
4.8
通讯作者：
Friedberg, Errol C.
中科院分区：
文献类型：
--
作者：
Guo, Caixia;Tang, Tie-Shan;Friedberg, Errol C.
Pol kappa蛋白是Y家族DNA聚合酶DinB/Pol kappa分支的一个真核生物成员，参与复制旁路对DNA损伤的耐受。尽管通过进化普遍保守，但Pol kappa在这一过程中的确切作用仍然未知。在这里，我们报告说，小鼠Pol κ蛋白可以物理相互作用的泛素酵母双杂交筛选，谷胱甘肽S-转移酶下拉，免疫沉淀方法。Pol κ与泛素的结合需要位于Pol κ C末端的泛素结合基序。此外，Pol κ与单泛素化增殖细胞核抗原（PCNA）的结合比与非泛素化PCNA的结合更强。泛素结合基序介导单泛素化的PCNA和Pol κ之间增强的关联。泛素结合基序也是Pol κ在UV辐射后形成核灶所必需的。然而，泛素结合基序不影响Pol κ半衰期。最后，我们研究了小鼠细胞暴露于苯并[a]芘-二氢二醇环氧化物或UVB辐射后的Pol κ表达水平。
Pol kappa protein is a eukaryotic member of the DinB/Pol kappa branch of the Y-family DNA polymerases, which are involved in the tolerance of DNA damage by replicative bypass. Despite universal conservation through evolution, the precise role(s) of Pol kappa in this process has remained unknown. Here we report that mouse Pol kappa can physically interact with ubiquitin by yeast two-hybrid screening, glutathione S-transferase pulldown, and immuno-precipitation methods. The association of Pol kappa with ubiquitin requires the ubiquitin-binding motifs located at the C terminus of Pol kappa. In addition, Pol kappa binds with monoubiquitinated proliferating cell nuclear antigen (PCNA) more robustly than with non-ubiquitinated PCNA. The ubiquitin-binding motifs mediate the enhanced association between monoubiquitinated PCNA and Pol kappa. The ubiquitin-binding motifs are also required for Pol kappa to form nuclear foci after UV radiation. However, the ubiquitin-binding motifs do not affect Pol kappa half-life. Finally, we have examined levels of Pol kappa expression following the exposure of mouse cells to benzo[a] pyrene-dihydrodiol epoxide or UVB radiation.