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.
The primary objective of the proposed studies is to investigate the role of protein degradation in DNA repair. Xeroderma pigmentosum and Cockayne Syndrome are well studied DNA repair defects for which there is no treatment or cure. Our studies could pave the way towards identification of candidate proteins and biochemical functions that can be targeted by therapeutic agents.
|Dang, Francis Wang; Chen, Li; Madura, Kiran (2016) Catalytically Active Proteasomes Function Predominantly in the Cytosol. J Biol Chem 291:18765-77|
|Liang, Ruei-Yue; Chen, Li; Ko, Bo-Ting et al. (2014) Rad23 interaction with the proteasome is regulated by phosphorylation of its ubiquitin-like (UbL) domain. J Mol Biol 426:4049-60|
|Chen, Li; Madura, Kiran (2014) Yeast importin-Î± (Srp1) performs distinct roles in the import of nuclear proteins and in targeting proteasomes to the nucleus. J Biol Chem 289:32339-52|
|Chen, Li; Madura, Kiran (2014) Degradation of specific nuclear proteins occurs in the cytoplasm in Saccharomyces cerevisiae. Genetics 197:193-7|