Pif1家族解旋機制的結(jié)構(gòu)基礎研究
發(fā)布時間：2019-01-11 08:38
【摘要】：Pif1蛋白是解旋酶SF1B超家族成員,能夠利用ATP水解提供的能量,對雙鏈 DNA(dsDNA),DNA/RNA 雜合體,G4DNA(G-quadruplexDNA)進行 5'→3'方向的解旋,是生物體內(nèi)非常重要的一類解旋酶。由于其結(jié)合底物的多樣性,Pif1參與了細胞中多種生物學過程調(diào)控。Pif1可以通過解鏈端粒酶模板RNA與端粒DNA,使端粒酶從端粒游離下來,從而調(diào)控端粒的長度。在復制及轉(zhuǎn)錄過程中,Pif1能夠迅速解開G4結(jié)構(gòu),維持基因組穩(wěn)定性。Pif1還參與了岡崎片段的成熟及DNA損傷修復的BIR(Break-induced replication)通路,而且Pif1在核糖體DNA(ribosomalDNA,rDNA)的復制過程及線粒體基因組穩(wěn)定性維持等方面也發(fā)揮了重要作用。本文主要利用大腸桿菌表達系統(tǒng),在體外分別構(gòu)建了釀酒酵母(Saccharomyces cerevisiae)ScPif1,人源hPif1,細菌(Bacteroides sp)BaPif1 全長及對應的解旋酶結(jié)構(gòu)域(Helicasedomain,HD)截短。利用X光晶體衍射技術(shù),解析了細菌BaPif1全長,人源hPif1-HD截短的單體結(jié)構(gòu),以及BaPif1與單鏈DNA dTI0,與雙鏈DNAdH的復合物晶體結(jié)構(gòu)。對比單體結(jié)構(gòu)與復合物結(jié)構(gòu)發(fā)現(xiàn)BaPif1結(jié)合DNA后發(fā)生了顯著的構(gòu)象變化,2A、2B結(jié)構(gòu)域分別發(fā)生了近20°、50°的旋轉(zhuǎn),并且這種構(gòu)象變化對于Pif1行使解旋酶功能是必須的;BaPif1與雙鏈DNAH復合物晶體結(jié)構(gòu)顯示結(jié)合的長鏈DNA在ssDNA/dsDN A junction處發(fā)生了近90°的彎曲,這有利于雙鏈解旋。接著結(jié)合生物化學、生物物理、分子與細胞生物學方法對解析的結(jié)構(gòu)進行深入分析,我們又闡述了 Pif1家族所特有的signature motif在解旋過程中所起的作用,并提出了可能的解旋模型。本文研究不僅為Pif1維持基因組穩(wěn)定性的作用提供重要依據(jù),而且為抗腫瘤新藥研究提供新的靶點和思路。
[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 擄and 50 擄, 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 擄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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q78;Q55
本文編號：2406919
[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 擄and 50 擄, 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 擄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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q78;Q55
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