| 研究課題/領域番号 |
14F04093
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| 研究機関 | 名古屋大学 |
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研究代表者 |
荻 朋男 名古屋大学, 環境医学研究所, 教授 (80508317)
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| 研究分担者 |
GUO Chaowan 名古屋大学, 環境医学研究所, 外国人特別研究員
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| 研究期間 (年度) |
2014-04-25 – 2017-03-31
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| キーワード | TC-NER / RNA polymerase II / Ubiquitination / UV sensitive syndrome / Cockyane syndrome |
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| 研究実績の概要 |
1. To understand the molecular mechanism of initiation of TC-NER that involve in the ubiquitination of RNA polIIo after UV damage, we treated cells with various reagents related to ubiquitination process and found that treatment of Reagent-A significantly abolished the UV specific ubiquitination of RNA polIIo. Reagent-A treated cells showed normal GG-NER repair activity but decreased TC-NER repair activity after UV damage. siRNA Knock down experiments further found a previously reported E3 ligase may involved in the RNA polIIo ubiquitination process, the exact mechanism is under investigation. We also uderwent reagent screening to further identify UVSSA-RNA polIIo interaction.
2. To get insights into UVSSA dependent RNA polIIo ubiquitination and initiation of TC-NER, we constructed several truncation mutants of GST-UVSSA and all proteins were successfully purified. GST-pull down experiments were performed to study the interaction of the UVSSA deletion proteins and TC-NER factors. We identified several domains that are important for protein-protein interactions.
3. By both in vitro and in vivo ubiquitination assay, we tried to identify ubiquitination sites of RNA polIIo. Expression of the mutant UVSSA in the UVSSA-deficient cell strains failed to rescue the RRS activity after UV irradation. Some UVSSA mutants are able to rescue the RNA polIIo ubiquitination in UV damaged UVSSA-deficient cells, but, the turnover/degradation of RNA polIIo in mutant UVSSA expressing cells are much faster than UVSSA wild type expressing cells.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We found a novel molecular process that may involve in the RNA pol II processing after UV DNA damage, and provided more insights into the biological significance of UVSSA in the initiation step of TC-NER.
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| 今後の研究の推進方策 |
1. In order to obtain further understanding of the molecular mechanism of UVSSA in the RNA pol IIo ubiquitnation, we had successfully established UVSSA knock out cell lines by CRISPR/Cas9 technology. We will setup a SILAC-based quantitative proteomics method for efficient isolation and enrichment of ubiquitinated peptides, in combination with high-resolution quantitative MS spectrometry. We will identify UVSSA-dependent ubiquitination of RNA polIIo upon UV damage. This is useful for us to generate non-ubiquitinable mutants to test biological relevance of the ubiquitination of RNA pol IIo on TC-NER.
2. Establishment of GFP-RBP1 stably expressing cells with various NER-factor-KO cell lines are also ongoing. Living cell imaging of GFP-RPB1 in combination with photobleaching experiments (FRAP&FLIP) will enables us to determine the dynamic kinetics of GFP-RBP1 in nuclear after UV damage and allow us to study which TCR proteins are involved in the initiation step of TC-NER and give valuable information on the reaction order of repair pathway.
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