Functional analysis of XAB2 complex involved in transcription and DNA repair

XAB2 复合物参与转录和 DNA 修复的功能分析

• 批准号: 13680763
• 负责人: NAKATSU Yoshimichi
• 金额: $ 2.3万
• 依托单位: Kyushu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680763/
• 关键词: nucreotide excision repair; transcription; TCR; XPA; XAB2; RNAポリメラーゼII; 遺伝子欠損マウス; CSA; CSB; 転写; PPIE

XPA-binding protein 2 (XAB2) is involved in transcription and transcription-coupled repair (TCR), and interacts with the TCR factors, CSA, CSB and RNA polymerase II. Flag-tagged XAB2 was expressed in HeLa cells and purified with ant-Flag antibody. Flag-tagged XAB2 was co-purified with about 20 polypeptides. Consistent with previous observation that microinjected anti-XAB2 antibody inhibits transcription and TCR, this affinity purified XAB2 complex enhanced FACT activity that stimulates an in vitro transcription elongation reaction using a chromatin template. Further purification of this protein complex with gel-filtration and miniQ column chromatography indicated that XAB2 was tightly associated with 5 polypeptides: p160, p57, p50, p42, and p35. Analysis using the mass spectrography revealed that p35 is peptidy1 proly1 cis-trans isomerase E (PPIE), a rotamase with RNA binding activity. Yeast 2-hybrid assay and in vitro pull-down assay with several deletion mutant proteins of PPIE indicated that XAB2 is directly associated with PPIE and RNA binding domain of PPIE is essential for the interaction.

XPA 结合蛋白 2 (XAB2) 参与转录和转录偶联修复 (TCR)，并与 TCR 因子、CSA、CSB 和 RNA 聚合酶 II 相互作用。带 Flag 标签的 XAB2 在 HeLa 细胞中表达，并用 ant-Flag 抗体纯化。 Flag 标记的 XAB2 与约 20 种多肽共纯化。与之前观察到的显微注射抗 XAB2 抗体抑制转录和 TCR 一致，这种亲和纯化的 XAB2 复合物增强了 FACT 活性，可使用染色质模板刺激体外转录延伸反应。通过凝胶过滤和 miniQ 柱色谱进一步纯化该蛋白质复合物表明 XAB2 与 5 种多肽紧密相关：p160、p57、p50、p42 和 p35。质谱分析表明，p35 是 peptidy1 proly1 cis-trans 异构酶 E (PPIE)，一种具有 RNA 结合活性的旋转异构酶。酵母2-杂交试验和体外pull-down试验表明，XAB2与PPIE直接相关，并且PPIE的RNA结合域对于这种相互作用至关重要。

项目成果

Tanaka K, Kamiuchi S, Ren Y, Yonemasu R, Ichikawa M, Murai H, Yoshino M, Yakeuchi S, Saijo M, Nakatsu Y, Miyauchi-Hashimoto H, Horio T.: "UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision rep

Tanaka K、Kamiuchi S、Ren Y、Yonemasu R、Ichikawa M、Murai H、Yoshino M、Yakeuchi S、Saijo M、Nakatsu Y、Miyauchi-Hashimoto H、Horio T.：“紫外线诱导的着色性干皮病 A 组皮肤癌变

Nakatsu Y.: "Transcription-coupled repair and Cockayne syndrome"Tanpakushitsu Kakusan Koso. 46. 908-915 (2001)

Nakatsu Y.：“转录偶联修复和科凯恩综合征”Tanpakushitsu Kakusan Koso。

Tanaka K, Kamiuchi S, Ren Y, Yonemasu R, Ichikawa M, Murai H, Yoshino M, Takeuchi S, Saijo M, Nakatau Y, Miyauchi-Hashimoto H, Horio T.: "UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision rep

Tanaka K、Kamiuchi S、Ren Y、Yonemasu R、Ichikawa M、Murai H、Yoshino M、Takeuchi S、Saijo M、Nakatau Y、Miyauchi-Hashimoto H、Horio T.：“紫外线诱导的着色性干皮病 A 组皮肤癌变

Murai M., Enokido Y., Inamura N., Yoshino M, Nakatsu Y., van der Horst GT., Hoeijmakers JH., Tanaka K., Hatanaka H.: "Early postnatal ataxja and abnormal cerebellar development in mice lacking Xeroderma pigmentosum Group A and Cockayne syndrome Group B DN

Murai M.、Enokido Y.、Inamura N.、Yoshino M、Nakatsu Y.、van der Horst GT.、Hoeijmakers JH.、Tanaka K.、Hatanaka H.：“缺乏着色性干皮病小鼠的早期产后共济失调和小脑发育异常

Yoshino M, Nakatsu Y, Riele H, Hirota S, Kitamura Y, Tanaka K.: "Additive roles of XPA and MSH2 genes in UVB-induced skin tumorigenesis in mice"DNA Repair. 1・11. 935-940 (2002)

Yoshino M、Nakatsu Y、Riele H、Hirota S、Kitamura Y、Tanaka K.：“XPA 和 MSH2 基因在 UVB 诱导的小鼠皮肤肿瘤发生中的附加作用”DNA Repair 1・11（2002）。