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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王惠鈞(Andrew H.-J. Wang) | |
dc.contributor.author | Li-Tzu Chen | en |
dc.contributor.author | 陳立慈 | zh_TW |
dc.date.accessioned | 2021-06-15T04:14:27Z | - |
dc.date.available | 2015-02-04 | |
dc.date.copyright | 2010-02-04 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-01-15 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45327 | - |
dc.description.abstract | 本論文分為兩個研究主題,第一是針對Sulfolobus solfataricus (Sso) 菌株中的同源重組蛋白酶 RadA 結構與功能的探討;第二個主題是根據大腸桿菌中同源重組蛋白酶RecA 和其去氧核醣核酸複合體結構,設計一系列的缩氨酸,其中一個可以明顯地增進同源重組蛋白酶重組去氧核醣核酸的效率。
去氧核醣核酸酶可以修補細胞內去氧核醣核酸的雙骨斷裂,這是一個演化上 保留的機制且普遍存在於細菌、古生菌和真核生物中。在過去的研究中,發現同 源重組蛋白酶主要會形成環型的聚合體,並在有單股去氧核醣核酸(雙股去氧核醣 核酸斷裂後,由核酸酶將雙股變成單股去氧核醣核酸)與三磷酸腺苷的情況下,會 與單股去氧核醣核酸形成右旋的絲狀複合體,這被認為是具有活性的同源重組蛋 白酶,然而此右旋的絲狀體複合體會尋找與其單股去氧核醣核酸同源的雙股去氧 核醣核酸,並以此雙股去氧核醣核酸為模板進行修補,完成同源重組。 使用原子力與電子顯微鏡,我們發現同源重組蛋白酶除了原先發現的環狀與 右旋狀的聚合體結構外,還存在另外兩種型式: 左旋絲狀與過度纏繞右旋的聚合 體。以X 光繞射的技術,我們成功地解出這兩種新的聚合體形式。經由一連串結 構的分析與比較此四種不同型式的同源重組蛋白酶聚合體(環狀、右旋、左旋和過 度纏繞右旋),我們建立一個假說: 同源重組蛋白酶先存在環狀複合體,在有單股 去氧核醣核酸時會變成具有酵素活性的右旋複合體,接著結構變化成過度纏繞右 旋的聚合體以利同源去氧核醣核酸之間的配對,最後以左旋複合體的形式將完成同源重組的產物移除,然後回到環狀結構。在此過程中同源重組蛋白酶可以三百 六十度旋轉已達到所需要的複合體結構變化。另外,我們也針對過度纏繞右旋的 聚合體是如何進行去氧核醣核酸之間的同源重組,做了一系列蛋白質點突變的分 析實驗。 在第二個主題中,我們設計了17 種不同序列的縮胺酸,其中特定的序列,例 如:IRFLTARRR 有能力促進同源同組蛋白酶的酵素活性,且在細胞的實驗中,能 保護細胞免於傷害去氧核醣核酸藥物的作用。我們發現帶正電、疏水性的胺基酸 與這些胺基酸之間的距離是能促進同源重組蛋白酶的必要因子,其作用的分子機 制也有深入的探討。然而這些縮胺酸的發現也有助於以後生物學上的應用。 | zh_TW |
dc.description.abstract | This thesis focused on two subjects. One is the study on structure and function of Sulfolobus solfataricus (Sso) RadA. Another is the peptide (IRFLTARRR) derived from
structure of RecA-DNA complex can promote not only the enzymatic activity of RecA protein but also resistance to DNA damaging agents. The RecA family of proteins is essential in homologous recombination, an evolutionarily conserved pathway that maintains genomic stability by protecting against DNA double strand breaks. In the previous reports, RecA family of proteins is thought to perform DNA strand exchange as a right-handed filament (active form) or as a closed-ring (inactive form). In this thesis, we report two new crystal structures that are left-handed and overwound right-handed helical filaments. Comparing the four different structures, we suppose that the DNA homology pairing and strand exchange occurs in the overwound right-handed nucleoprotein filament, and release of DNA exchange final products using the left-handed filament. We also identified the conserved hinge region (subunit rotation motif) in which a 360° clockwise axial rotation accompanies stepwise structural transitions from a closed ring to the right-handed filament, then to an overwound right-handed filament and finally to the left-handed filament. The results of several in vitro experiments are consistent with our hypothesis. Another story is about a rationally-designed small peptide based on the Escherichia coli RecA-DNA crystal structure can promote homologous recombination through the enhancement of both RecA-mediated strand assimilation and three-strand exchange activity. We identified that the hydrophobicity and poly-positive charges, and the space between them in those small peptides are crucial features for such activities. Remarkably, peptide #3 alone without RecA can also promote the D-loop formation at elevated temperature. Cell viability assays showed that the peptide elevates mammalian cell resistance to two cytotoxic DNA drugs, cisplatin and doxorubicin. The rescue of viability may result from increased DNA repair efficiency. Such peptides may find future biological applications. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T04:14:27Z (GMT). No. of bitstreams: 1 ntu-99-F93b46007-1.pdf: 8305301 bytes, checksum: dc7100d228761b5e8a0f60450e3075bc (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 摘要…………………………………………………………………………i
Abstract…………………………………………………………………….ii CHAPTER 1 INTRODUCTION…………………………………………...1 CHAPTER 2 MATERIALS AND METHODS…………………………….6 Protein expression and purification……………………………………...6 Crystallization, data collection and structural determination……………7 DNA binding, ssDNA-stimulated ATPase activity and the strand assimilation assay………………………………………………………9 Surface plasmon resonance (SPR)……………………………….…….10 Double-strand DNA binding assay………………………..………….11 E.coli RecA Proteins and peptides……………………………………12 Three-strand exchange assay…………………………………………12 Electrophoretic mobility shift assay (EMSA) for the peptide #3………13 Cell survival assay………………………………………….…………14 CHAPTER 3 RESULTS……………………………………..……………15 Part A: crystal structures of SsoRadA left- and overwound right-handed helical filaments…………………………………………………………15 Observation of SsoRadA left- and right-handed helical filaments byatomic force microscopy (AFM) and electronic microscopy (EM)………………………………………………………………….15 Crystal structures of the SsoRadA left- and right-handed helical filaments………………………………………………………………16 Comparing the RadA quaternary structures of closed ring, right-handed, overwound right-handed and left-handed conformations……………18 Discovery subunit rotation motif (SRM)………………………………18 Subunit rotation influences the protomer-protomer interface for DNA and ATP binding………………………………………………………19 SRM is crucial for SsoRadA function………………………………22 Spatial arrangement of the L1 loop and N-terminal domain in the overwound right-handed SsoRadA filament…………………………24 Structure of the L1 loop……………………………………………..….25 Structure of the N-terminal domain…………………………………..26 R217, R223 and R229 are critical for single-strand DNA binding…..28 K27 and K60 are specifically important for double-stranded DNA binding…………………………………………………………………29 K27, K60, R217, R223 and R229 are all essential for the D-loopformation……………………………………………………………31 Part B: A rationally designed peptide enhances homologous recombination……………………………………………………………32 Rationally designed peptides based on RecA-ssDNA structure………32 Peptide #3 stimulates the RecA-mediated strand assimilation…………33 Peptide #3 stimulates RecA activities by a mechanism that does not influence the ATP hydrolysis…………………………………………37 Peptide #3 alone can induce strand assimilation activity………………39 Peptide #3 promotes resistance toward DNA damaging agents………42 CHAPTER 4 DISCUSSION……………………………………………45 REFERENCES…………………………………………………………50 FIGURES…………………………………………………………………57 Figure 1. Visualization of the SsoRadA protein filaments using AFM with the carbon nanotube tip method…………………………………58 Figure 2. Visualization of the SsoRadA protein filaments using EM…59 Figure 3. Crystal structures of left-handed and overwound right-handed SsoRadA filaments……………………………………………………60 Figure 4. Sequence alignment of RecA family proteins………………61 Figure 5. Quaternary structures of SsoRadA (the left-handed andoverwound right-handed forms), MvRadA-AMP-PNP and PfRad51…62 Figure 6. Comparing these monomer structures of MvRadA-AMP-PNP, PfRad51 and SsoRadA (the left-handed and overwound right-handed forms)………………………………………………………………63 Figure 7. Superposition of MvRadA-AMP-PNP, PfRad51 and SsoRadA (the left-handed and overwound right-handed forms)………………64 Figure 8. E1-R0-E2 triad influences ATP and DNA binding…………65 Figure 9. SRM is crucial for SsoRadA D-loop formation……………66 Figure 10. SRM influences SsoRadA ATP hydrolysis and single-strand DNA binding………………………………………………………67 Figure 11. Spatial arrangement of the L1 motif and the (HhH)2 domain along a overwound SsoRadA right-handed filament…………………68 Figure 12. The architecture and structure of the L1 single-stranded DNA binding loop…………………………………………………………….69 Figure 13. The architecture and structure of the (HhH)2 domain………70 Figure 14. Real time interaction of the L1 binding loop and (HhH)2 domain mutant SsoRadA proteins to single-stranded DNA substrate analyzed using a Biacore X instrument………………………………71 Figure 15. EMSA analysis for double-stranded DNA binding………72 Figure 16. K27, K60, R217, R223 and R229 are all essential for the D-loop formation………………………………………………………73 Figure 17. The structure of E.coli RecA and a single-stranded DNA complex………………………………………………………………74 Figure 18. Peptide #3 enhances the strand assimilation activity due to its composition of positively charged amino acids following the hydrophobic amino acids……………………………………………75 Figure 19. Peptide #3 can enhance both RecA-mediated strand assimilation and three-strand exchange………………………………77 Figure 20. Peptide #3 alone expresses strong strand assimilation activity due to its preference for the D-loop or D-loop mimic structure……….79 Figure 21. Peptide #3 rescues the viability of A375 and MCF-7 cells after treatment with DNA damaging agents……………………………80 Figure 22. E. coli RecA proteins may also use the similar SRM to control the structural transition…………………………………………………81 Figure 23. The rotary motor hypothesis for RadA protein filaments…82 Figure 24. A hypothesis for RadA-mediated homology pairing……83 TABLES…………………………………………………………………84 Table I. Data collection and refinement statistics for the RadA P43crystal…………………………………………………………………85 Table II. Data collection and refinement statistics for the RadA P31 crystal…………………………………………………………………86 LIST OF PUBLICATION……………………………………………87 Published Papers…………………………………………………………88 | |
dc.language.iso | en | |
dc.title | 同源重組蛋白酶的結構功能分析與合理設計之縮氨酸可調控同源重組蛋白酶 | zh_TW |
dc.title | Structural and Functional Analysis of RecA-like Recombinases and Rationally-designed Peptides That Modulate the RecA Activities | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 蕭傳鐙(Chwan-Deng Hsiao),馬徹(Che Ma),陳光超(Guang-Chao Chen),史有伶(Yu-Ling Shih) | |
dc.subject.keyword | 同源重組,同源重組蛋白酶,縮胺酸,去氧核醣核酸,左旋絲狀體,X 光繞射,結構, | zh_TW |
dc.subject.keyword | RecA,homologous recombination,DNA repair,left-handed filaments,small peptides,drug design and strand exchange, | en |
dc.relation.page | 88 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-01-18 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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