A Role for Protein Degradation in Nucleotide Excision-Repair

描述(由申请人提供)：许多DNA修复因子也在泛素/蛋白酶体系统中发挥作用，这是一种消除重要调节因子的保守机制。值得注意的例子包括参与激活Fanconi贫血(FA)途径的FANC-L，以及调节DNA损伤诱导的信号系统的BRCA1和BRCA2蛋白。这些因素分别将蛋白质降解机制与DNA交联链和双链断裂的修复联系起来。然而，尽管进行了广泛的研究，但蛋白质周转在这些修复机制中的重要性还没有被很好地理解。其他在DNA修复和蛋白质降解中具有良好特征的蛋白质包括泛素结合酶Rad6(也称为Ubc2)。参与转录偶联修复(CsA；CSB)、核苷酸切除修复(RAD23；RAD7；RAD16；XPC；RAD4)和转录(RNAPol II)的因子也与蛋白质降解途径相交，或者是蛋白酶体介导的降解靶标。被蛋白酶体降解的关键调控因子几乎完全是核蛋白。这些因素的周转已经被详尽地研究过，尽管还没有系统的努力来确定核蛋白在哪里被降解。虽然在细胞核中检测到蛋白酶体亚单位，但没有证据表明完整的、具有催化活性的蛋白酶体在细胞核中进行蛋白分解。我们最近报道，酵母菌中的Sts1蛋白将蛋白酶体定位于核周。Sts1的突变完全阻断了蛋白酶体的核定位。我们确定Rad4和其他底物在sts1-2中是稳定的。值得注意的是，核蛋白在核出口突变体中也是稳定的，这为我们的假设提供了令人信服的支持，即许多(如果不是大多数)核底物被出口到靠近核外围的胞浆蛋白酶体。同样值得注意的是，sts1-2和出口突变体中的稳定蛋白都积累在细胞核中。我们发现，RAD4 DNA修复蛋白被蛋白酶体迅速降解。建议进一步研究蛋白质降解在核苷酸切除-修复(NER)中的作用。为了确定其降解的特征，我们检查了sts1-2中的Rad4水平，发现它是稳定的，因为蛋白酶体在这个突变体中不是针对核的。值得注意的是，Rad4在多个核输出突变体中也是稳定的，这表明它的降解需要输出到胞浆蛋白酶体。这些发现意义重大，因为它们暗示核出口机制在促进一种关键DNA修复因子的降解方面发挥了新的重要作用。我们将测试核输出和蛋白酶体靶向机制中突变对Rad4稳定性和核苷酸切除修复的影响。我们将具体描述RAD4的输出以及胞质蛋白酶体对其的降解。切除环丁烷嘧啶二聚体(CPD)和6-4光产物(6-4 PPS)，以及DNA修复完成后重新进入细胞周期，将在核出口和蛋白酶体靶向突变体中进行测试。补充研究将使用经典的T4内切酶V裂解试验来测量紫外线诱导DNA损伤后T^T DNA损伤的清除情况。

DESCRIPTION (provided by applicant): Many DNA repair factors also play a role in the ubiquitin/proteasome system, which is a conserved mechanism for eliminating important regulatory factors. Notable examples include FANC-L which is implicated in the activation of the Fanconi anemia (FA) pathway, and BRCA1 and BRCA2 proteins, which regulate a DNA damage-induced signaling system. These factors link protein degradation mechanisms to the repair of DNA cross-links and double strand breaks, respectively. However, despite extensive investigation, the significance of protein turnover in these repair mechanisms is not well understood. Other proteins with well-characterized roles in both DNA repair and protein degradation include the ubiquitin conjugating-enzyme Rad6 (also known as Ubc2). Factors involved in transcription-coupled repair (CSA; CSB), nucleotide excision repair (Rad23; Rad7; Rad16; XPC; Rad4) and transcription (RNA Pol II) also intersect with the protein degradation pathway, or are targets of proteasome-mediated degradation. Critical regulatory factors that are degraded by the proteasome are almost exclusively nuclear proteins. The turnover of these factors has been studied exhaustively, although there has been no systematic effort to determine where nuclear proteins are degraded. Whereas proteasome subunits are detected in the nucleus, there is no evidence that intact, catalytically active proteasomes carry out proteolysis in the nucleus. We reported recently that proteasomes are targeted to the nuclear periphery by the Sts1 protein in yeast. Mutations in Sts1 completely blocked nuclear localization of proteasomes. We determined that Rad4 and other substrates were stabilized in sts1-2. Remarkably, nuclear proteins were also stabilized in nuclear export mutants, providing compelling support for our hypothesis that many, if not most, nuclear substrates are exported to cytosolic proteasomes near the nuclear periphery. It is also noteworthy that the stabilized proteins in both sts1-2 and export mutants accumulated in the nucleus. We found that the Rad4 DNA repair protein is rapidly degraded by the proteasome. Studies are proposed to further examine the role of protein degradation in nucleotide excision-repair (NER). In an effort to characterize its degradation we examined Rad4 levels in sts1-2 and found that it was stabilized, because proteasomes are not targeted to the nucleus in this mutant. Remarkably, Rad4 was also stabilized in multiple nuclear export mutants, demonstrating that its degradation requires export to cytosolic proteasomes. These findings are significant because they implicate a new and important role for the nuclear export mechanism in promoting the degradation of a key DNA repair factor. We will test the effect of mutations in the nuclear export and proteasome-targeting mechanisms on Rad4 stability and nucleotide excision repair. We will specifically characterize the export of Rad4 and its degradation by cytosolic proteasomes. The excision of cyclobutane pyrimidine dimers (CPDs) and 6- 4 photoproducts (6-4 PPs), and the reentry into the cell cycle upon completion of DNA repair will be tested in nuclear export and proteasome-targeting mutants. Complementary studies will use the classic T4 endonuclease V cleavage assay to measure the removal of T^T DNA lesions following UV-induced DNA damage.