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Pif1家族解旋机制的结构基础研究

发布时间:2019-01-11 08:38  文章来源:笔耕文化传播
【摘要】:Pif1蛋白是解旋酶SF1B超家族成员,能够利用ATP水解提供的能量,对双链 DNA(dsDNA),DNA/RNA 杂合体,G4DNA(G-quadruplexDNA)进行 5'→3'方向的解旋,是生物体内非常重要的一类解旋酶。由于其结合底物的多样性,Pif1参与了细胞中多种生物学过程调控。Pif1可以通过解链端粒酶模板RNA与端粒DNA,使端粒酶从端粒游离下来,从而调控端粒的长度。在复制及转录过程中,Pif1能够迅速解开G4结构,维持基因组稳定性。Pif1还参与了冈崎片段的成熟及DNA损伤修复的BIR(Break-induced replication)通路,而且Pif1在核糖体DNA(ribosomalDNA,rDNA)的复制过程及线粒体基因组稳定性维持等方面也发挥了重要作用。本文主要利用大肠杆菌表达系统,在体外分别构建了酿酒酵母(Saccharomyces cerevisiae)ScPif1,人源hPif1,细菌(Bacteroides sp)BaPif1 全长及对应的解旋酶结构域(Helicasedomain,HD)截短。利用X光晶体衍射技术,解析了细菌BaPif1全长,人源hPif1-HD截短的单体结构,以及BaPif1与单链DNA dTI0,与双链DNAdH的复合物晶体结构。对比单体结构与复合物结构发现BaPif1结合DNA后发生了显著的构象变化,2A、2B结构域分别发生了近20°、50°的旋转,并且这种构象变化对于Pif1行使解旋酶功能是必须的;BaPif1与双链DNAH复合物晶体结构显示结合的长链DNA在ssDNA/dsDN A junction处发生了近90°的弯曲,这有利于双链解旋。接着结合生物化学、生物物理、分子与细胞生物学方法对解析的结构进行深入分析,我们又阐述了 Pif1家族所特有的signature motif在解旋过程中所起的作用,并提出了可能的解旋模型。本文研究不仅为Pif1维持基因组稳定性的作用提供重要依据,而且为抗肿瘤新药研究提供新的靶点和思路。
[Abstract]:Pif1 protein is a member of the SF1B superfamily of helicases. It can use the energy provided by ATP hydrolysis to unspin the double-stranded DNA (dsDNA), DNA/RNA heterozyme, G4DNA (G-quadruplexDNA). It is a very important kind of helicase in organism. Because of the diversity of its binding substrates, Pif1 participates in many biological processes in cells. Pif1 can free telomerase from telomere by unchaining telomerase template RNA and telomere DNA, thereby regulating the length of telomere. In the process of replication and transcription, Pif1 can rapidly unravel G4 structure and maintain genomic stability. Pif1 is also involved in the BIR (Break-induced replication) pathway of Okazaki fragment maturation and DNA damage repair, and Pif1 plays an important role in ribosomal DNA (ribosomalDNA,. The replication process of rDNA and the maintenance of mitochondrial genome stability also play an important role. In this paper, the full-length (Bacteroides sp) BaPif1 of human hPif1, bacteria of Saccharomyces cerevisiae (Saccharomyces cerevisiae) ScPif1,) and the truncation of the corresponding helicase domain (Helicasedomain,HD) were constructed in vitro by using Escherichia coli expression system. X-ray crystal diffraction technique was used to analyze the full length of bacterial BaPif1, the truncated monomer structure of human hPif1-HD, and the crystal structure of the complex of BaPif1 with single-stranded DNA dTI0, and double-stranded DNAdH. Comparing the monomer structure with the complex structure, it was found that the conformation of BaPif1 combined with DNA changed significantly, the 2A2B domain rotated nearly 20 掳and 50 掳, respectively, and this conformation change was necessary for Pif1 to perform the function of helicase. The crystal structure of BaPif1 and double-stranded DNAH complex shows that the combined long-stranded DNA bends nearly 90 掳at ssDNA/dsDN A junction, which is favorable to double strand unspin. Then the structure of the solution was analyzed by biochemistry, biophysics, molecular and cell biology, and the role of signature motif, which is unique to the Pif1 family, in the process of unspin was discussed, and the possible model of the solution was proposed. This study not only provides an important basis for Pif1 to maintain genomic stability, but also provides new targets and ideas for the study of new antitumor drugs.
【学位授予单位】:浙江大学
【学位级别】:博士
【学位授予年份】:2017
【分类号】:Q78;Q55

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