A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway
A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway
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DOI：
10.1016/j.molcel.2007.09.031
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发表时间：
2007-12-14
期刊：
影响因子：
16
通讯作者：
D'Andrea, Alan D.
中科院分区：
文献类型：
--
作者：
Cohn, Martin A.;Kowal, Przemyslaw;D'Andrea, Alan D.
去泛素化酶USP 1控制DNA损伤反应蛋白Ub-FANCD 2的细胞水平，Ub-FANCD 2是范可尼贫血DNA修复途径的关键蛋白。在这里，我们报告了纯化的USP 1多亚基蛋白复合物从HeLa细胞含有化学计量的WD 40重复含有蛋白质，USP 1相关因子1（UAF 1）。使用泛素-7-氨基-4-甲基香豆素（Ub-AMC）或纯化的单泛素化FANCD 2蛋白作为底物，体外重建USP 1去泛素化酶活性，证明LIAR作为USP 1的激活剂发挥作用。UAF 1结合增加了催化转换（k（cat）），但不增加USP 1酶对底物的亲和力（K-M）。此外，我们发现DNA损伤导致USP 1基因的转录立即关闭，导致USP 1/UAF 1蛋白复合物迅速下降。总之，我们的研究结果描述了去泛素化酶，USP 1，和DNA修复的调节机制。
The deubiquitinating enzyme USP1 controls the cellular levels of the DNA damage response protein Ub-FANCD2, a key protein of the Fanconi anemia DNA repair pathway. Here we report the purification of a USP1 multisubunit protein complex from HeLa cells containing stoichiometric amounts of a WD40 repeat-containing protein, USP1 associated factor 1 (UAF1). In vitro reconstitution of USP1 deubiquitinating enzyme activity, using either ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) or purified monoubiquitinated FANCD2 protein as substrates, demonstrates that LIAR functions as an activator of USP1. UAF1 binding increases the catalytic turnover (k(cat)) but does not increase the affinity of the USP1 enzyme for the substrate (K-M). Moreover, we show that DNA damage results in an immediate shutoff of transcription of the USP1 gene, leading to a rapid decline in the USP1/UAF1 protein complex. Taken together, our results describe a mechanism of regulation of the deubiquitinating enzyme, USP1, and of DNA repair.