請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/45327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 | |
| dc.identifier.citation | 1. Clark, A.J. and Margulies, A.D. (1965) Isolation and characterization of
recombination-deficient mutants of Escherichia Coli K12. Proc Natl Acad Sci U S A, 53, 451-459. 2. Sandler, S.J., Satin, L.H., Samra, H.S. and Clark, A.J. (1996) recA-like genes from three archaean species with putative protein products similar to Rad51 and Dmc1 proteins of the yeast Saccharomyces cerevisiae. Nucleic Acids Res, 24, 2125-2132. 3. Shinohara, A., Ogawa, H. and Ogawa, T. (1992) Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein. Cell, 69, 457-470. 4. Bishop, D.K., Park, D., Xu, L. and Kleckner, N. (1992) DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell, 69, 439-456. 5. Cox, M.M. (2003) The bacterial RecA protein as a motor protein. Annu Rev Microbiol, 57, 551-577. 6. West, S.C. (2003) Molecular views of recombination proteins and their control. Nat Rev Mol Cell Biol, 4, 435-445. 7. Sung, P. and Klein, H. (2006) Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol, 7, 739-750. 8. San Filippo, J., Sung, P. and Klein, H. (2008) Mechanism of eukaryotic homologous recombination. Annu Rev Biochem, 77, 229-257. 9. Bianco, P.R., Tracy, R.B. and Kowalczykowski, S.C. (1998) DNA strand exchange proteins: a biochemical and physical comparison. Front Biosci, 3, D570-603. 10. Story, R.M., Weber, I.T. and Steitz, T.A. (1992) The structure of the E. coli recA protein monomer and polymer. Nature, 355, 318-325. 11. Chen, Z., Yang, H. and Pavletich, N.P. (2008) Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structures. Nature, 453, 489-484. 12. Pellegrini, L., Yu, D.S., Lo, T., Anand, S., Lee, M., Blundell, T.L. and Venkitaraman, A.R. (2002) Insights into DNA recombination from the structure of a RAD51-BRCA2 complex. Nature, 420, 287-293. 13. Aihara, H., Ito, Y., Kurumizaka, H., Yokoyama, S. and Shibata, T. (1999) The N-terminal domain of the human Rad51 protein binds DNA: structure and a DNA binding surface as revealed by NMR. J Mol Biol, 290, 495-504. 14. Kinebuchi, T., Kagawa, W., Kurumizaka, H. and Yokoyama, S. (2005) Role ofthe N-terminal domain of the human DMC1 protein in octamer formation and DNA binding. J Biol Chem, 280, 28382-28387. 15. McIlwraith, M.J., Hall, D.R., Stasiak, A.Z., Stasiak, A., Wigley, D.B. and West, S.C. (2001) RadA protein from Archaeoglobus fulgidus forms rings, nucleoprotein filaments and catalyses homologous recombination. Nucleic Acids Res, 29, 4509-4517. 16. Wu, Y., He, Y., Moya, I.A., Qian, X. and Luo, Y. (2004) Crystal structure of archaeal recombinase RADA: a snapshot of its extended conformation. Mol Cell, 15, 423-435. 17. Ariza, A., Richard, D.J., White, M.F. and Bond, C.S. (2005) Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA. Nucleic Acids Res, 33, 1465-1473. 18. Yang, S., Yu, X., Seitz, E.M., Kowalczykowski, S.C. and Egelman, E.H. (2001) Archaeal RadA protein binds DNA as both helical filaments and octameric rings. J Mol Biol, 314, 1077-1085. 19. Lee, M.H., Leng, C.H., Chang, Y.C., Chou, C.C., Chen, Y.K., Hsu, F.F., Chang, C.S., Wang, A.H. and Wang, T.F. (2004) Self-polymerization of archaeal RadA protein into long and fine helical filaments. Biochem Biophys Res Commun, 323, 845-851. 20. Shin, D.S., Pellegrini, L., Daniels, D.S., Yelent, B., Craig, L., Bates, D., Yu, D.S., Shivji, M.K., Hitomi, C., Arvai, A.S. et al. (2003) Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2. Embo J, 22, 4566-4576. 21. Kinebuchi, T., Kagawa, W., Enomoto, R., Tanaka, K., Miyagawa, K., Shibata, T., Kurumizaka, H. and Yokoyama, S. (2004) Structural basis for octameric ring formation and DNA interaction of the human homologous-pairing protein Dmc1. Mol Cell, 14, 363-374. 22. Conway, A.B., Lynch, T.W., Zhang, Y., Fortin, G.S., Fung, C.W., Symington, L.S. and Rice, P.A. (2004) Crystal structure of a Rad51 filament. Nat Struct Mol Biol, 11, 791-796. 23. Qian, X., Wu, Y., He, Y. and Luo, Y. (2005) Crystal structure of Methanococcus voltae RadA in complex with ADP: hydrolysis-induced conformational change. Biochemistry, 44, 13753-13761. 24. Chang, Y.C., Lo, Y.H., Lee, M.H., Leng, C.H., Hu, S.M., Chang, C.S. and Wang, T.F. (2005) Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex. Biochemistry, 44, 6052-6058. 25. Lee, M.H., Chang, Y.C., Hong, E.L., Grubb, J., Chang, C.S., Bishop, D.K. and Wang, T.F. (2005) Calcium ion promotes yeast Dmc1 activity via formation oflong and fine helical filaments with single-stranded DNA. J Biol Chem, 280, 40980-40984. 26. Bugreev, D.V., Golub, E.I., Stasiak, A.Z., Stasiak, A. and Mazin, A.V. (2005) Activation of human meiosis-specific recombinase Dmc1 by Ca2+. J Biol Chem, 280, 26886-26895. 27. Sehorn, M.G., Sigurdsson, S., Bussen, W., Unger, V.M. and Sung, P. (2004) Human meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand exchange. Nature, 429, 433-437. 28. Thorslund, T. and West, S.C. (2007) BRCA2: a universal recombinase regulator. Oncogene, 26, 7720-7730. 29. Starita, L.M. and Parvin, J.D. (2003) The multiple nuclear functions of BRCA1: transcription, ubiquitination and DNA repair. Curr Opin Cell Biol, 15, 345-350. 30. Sung, P. (1997) Yeast Rad55 and Rad57 proteins form a heterodimer that functions with replication protein A to promote DNA strand exchange by Rad51 recombinase. Genes Dev, 11, 1111-1121. 31. Klein, H.L. (2008) The consequences of Rad51 overexpression for normal and tumor cells. DNA Repair (Amst), 7, 686-693. 32. Kawabata, M., Kawabata, T. and Nishibori, M. (2005) Role of recA/RAD51 family proteins in mammals. Acta Med Okayama, 59, 1-9. 33. Takata, M., Sasaki, M.S., Tachiiri, S., Fukushima, T., Sonoda, E., Schild, D., Thompson, L.H. and Takeda, S. (2001) Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol Cell Biol, 21, 2858-2866. 34. Hatanaka, A., Yamazoe, M., Sale, J.E., Takata, M., Yamamoto, K., Kitao, H., Sonoda, E., Kikuchi, K., Yonetani, Y. and Takeda, S. (2005) Similar effects of Brca2 truncation and Rad51 paralog deficiency on immunoglobulin V gene diversification in DT40 cells support an early role for Rad51 paralogs in homologous recombination. Mol Cell Biol, 25, 1124-1134. 35. Martin, R.W., Orelli, B.J., Yamazoe, M., Minn, A.J., Takeda, S. and Bishop, D.K. (2007) RAD51 up-regulation bypasses BRCA1 function and is a common feature of BRCA1-deficient breast tumors. Cancer Res, 67, 9658-9665. 36. Hays, S.L., Firmenich, A.A. and Berg, P. (1995) Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins. Proc Natl Acad Sci U S A, 92, 6925-6929. 37. Jayathilaka, K., Sheridan, S.D., Bold, T.D., Bochenska, K., Logan, H.L., Weichselbaum, R.R., Bishop, D.K. and Connell, P.P. (2008) A chemical compound that stimulates the human homologous recombination protein RAD51. Proc Natl Acad Sci U S A, 105, 15848-15853.38. Kim, H.K., Morimatsu, K., Norden, B., Ardhammar, M. and Takahashi, M. (2002) ADP stabilizes the human Rad51-single stranded DNA complex and promotes its DNA annealing activity. Genes Cells, 7, 1125-1134. 39. Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S. et al. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr, 54, 905-921. 40. Jones, T.A., Zou, J.Y., Cowan, S.W. and Kjeldgaard, M. (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A, 47 ( Pt 2), 110-119. 41. Chen, Y.K., Leng, C.H., Olivares, H., Lee, M.H., Chang, Y.C., Kung, W.M., Ti, S.C., Lo, Y.H., Wang, A.H., Chang, C.S. et al. (2004) Heterodimeric complexes of Hop2 and Mnd1 function with Dmc1 to promote meiotic homolog juxtaposition and strand assimilation. Proc Natl Acad Sci U S A, 101, 10572-10577. 42. Zhang, X.P., Lee, K.I., Solinger, J.A., Kiianitsa, K. and Heyer, W.D. (2005) Gly-103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 protein is critical for DNA binding. J Biol Chem, 280, 26303-26311. 43. LaPensee, E.W., Schwemberger, S.J., LaPensee, C.R., Bahassi el, M., Afton, S.E. and Ben-Jonathan, N. (2009) Prolactin confers resistance against cisplatin in breast cancer cells by activating glutathione-S-transferase. Carcinogenesis, 30, 1298-1304. 44. Seitz, E.M., Brockman, J.P., Sandler, S.J., Clark, A.J. and Kowalczykowski, S.C. (1998) RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange. Genes Dev, 12, 1248-1253. 45. Chen, L.T., Ko, T.P., Chang, Y.W., Lin, K.A., Wang, A.H. and Wang, T.F. (2007) Structural and functional analyses of five conserved positively charged residues in the L1 and N-terminal DNA binding motifs of archaeal RADA protein. PLoS One, 2, e858. 46. Wu, Y., Qian, X., He, Y., Moya, I.A. and Luo, Y. (2005) Crystal structure of an ATPase-active form of Rad51 homolog from Methanococcus voltae. Insights into potassium dependence. J Biol Chem, 280, 722-728. 47. Chen, L.T., Ko, T.P., Chang, Y.C., Lin, K.A., Chang, C.S., Wang, A.H. and Wang, T.F. (2007) Crystal structure of the left-handed archaeal RadA helical filament: identification of a functional motif for controlling quaternary structures and enzymatic functions of RecA family proteins. Nucleic Acids Res, 35, 1787-1801. 48. De Zutter, J.K. and Knight, K.L. (1999) The hRad51 and RecA proteins showsignificant differences in cooperative binding to single-stranded DNA. J Mol Biol, 293, 769-780. 49. Shao, X. and Grishin, N.V. (2000) Common fold in helix-hairpin-helix proteins. Nucleic Acids Res, 28, 2643-2650. 50. Pelletier, H., Sawaya, M.R., Kumar, A., Wilson, S.H. and Kraut, J. (1994) Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science, 264, 1891-1903. 51. Hollis, T., Ichikawa, Y. and Ellenberger, T. (2000) DNA bending and a flip-out mechanism for base excision by the helix-hairpin-helix DNA glycosylase, Escherichia coli AlkA. Embo J, 19, 758-766. 52. Chen, L., Haushalter, K.A., Lieber, C.M. and Verdine, G.L. (2002) Direct visualization of a DNA glycosylase searching for damage. Chem Biol, 9, 345-350. 53. Shim, K.S., Schmutte, C., Yoder, K. and Fishel, R. (2006) Defining the salt effect on human RAD51 activities. DNA Repair (Amst), 5, 718-730. 54. Lee, C.D. and Wang, T.F. (2009) The N-terminal domain of Escherichia coli RecA have multiple functions in promoting homologous recombination. J Biomed Sci, 16, 37. 55. Bugreev, D.V. and Mazin, A.V. (2004) Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc Natl Acad Sci U S A, 101, 9988-9993. 56. Petalcorin, M.I., Galkin, V.E., Yu, X., Egelman, E.H. and Boulton, S.J. (2007) Stabilization of RAD-51-DNA filaments via an interaction domain in Caenorhabditis elegans BRCA2. Proc Natl Acad Sci U S A, 104, 8299-8304. 57. Esashi, F., Galkin, V.E., Yu, X., Egelman, E.H. and West, S.C. (2007) Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2. Nat Struct Mol Biol, 14, 468-474. 58. Xing, X. and Bell, C.E. (2004) Crystal structures of Escherichia coli RecA in a compressed helical filament. J Mol Biol, 342, 1471-1485. 59. Xing, X. and Bell, C.E. (2004) Crystal structures of Escherichia coli RecA in complex with MgADP and MnAMP-PNP. Biochemistry, 43, 16142-16152. 60. Chang, Y.W., Ko, T.P., Lee, C.D., Chang, Y.C., Lin, K.A., Chang, C.S., Wang, A.H. and Wang, T.F. (2009) Three new structures of left-handed RADA helical filaments: structural flexibility of N-terminal domain is critical for recombinase activity. PLoS One, 4, e4890. 61. Modesti, M., Budzowska, M., Baldeyron, C., Demmers, J.A., Ghirlando, R. and Kanaar, R. (2007) RAD51AP1 is a structure-specific DNA binding protein that stimulates joint molecule formation during RAD51-mediated homologousrecombination. Mol Cell, 28, 468-481. 62. Vazquez, O., Blanco-Canosa, J.B., Vazquez, M.E., Martinez-Costas, J., Castedo, L. and Mascarenas, J.L. (2008) Efficient DNA binding and nuclear uptake by distamycin derivatives conjugated to octa-arginine sequences. Chembiochem, 9, 2822-2829. 63. Patel, L.N., Zaro, J.L. and Shen, W.C. (2007) Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives. Pharm Res, 24, 1977-1992. 64. Menz, R.I., Walker, J.E. and Leslie, A.G. (2001) Structure of bovine mitochondrial F(1)-ATPase with nucleotide bound to all three catalytic sites: implications for the mechanism of rotary catalysis. Cell, 106, 331-341. 65. Menetski, J.P. and Kowalczykowski, S.C. (1985) Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors. J Mol Biol, 181, 281-295. 66. Story, R.M. and Steitz, T.A. (1992) Structure of the recA protein-ADP complex. Nature, 355, 374-376. 67. Chi, P., San Filippo, J., Sehorn, M.G., Petukhova, G.V. and Sung, P. (2007) Bipartite stimulatory action of the Hop2-Mnd1 complex on the Rad51 recombinase. Genes Dev, 21, 1747-1757. 68. Wiese, C., Dray, E., Groesser, T., San Filippo, J., Shi, I., Collins, D.W., Tsai, M.S., Williams, G.J., Rydberg, B., Sung, P. et al. (2007) Promotion of homologous recombination and genomic stability by RAD51AP1 via RAD51 recombinase enhancement. Mol Cell, 28, 482-490. 69. Sugiyama, T., Kittaka, A., Takayama, H., Tomioka, M., Ida, Y. and Kuroda, R. (2003) Aggregation of RecA-derived peptides on single-stranded oligonucleotides triggered by schiff base-mediated crosslinking. Bioorg Med Chem Lett, 13, 2847-2851. 70. Sugiyama, T., Kittaka, A., Takayama, H., Tomioka, M., Ida, Y. and Kuroda, R. (2000) Interaction of peptides derived from RecA with single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine. Nucleic Acids Symp Ser, 41-42. 71. Voloshin, O.N., Wang, L. and Camerini-Otero, R.D. (1996) Homologous DNA pairing promoted by a 20-amino acid peptide derived from RecA. Science, 272, 868-872. 72. Nishinaka, T., Ito, Y., Yokoyama, S. and Shibata, T. (1997) An extended DNA structure through deoxyribose-base stacking induced by RecA protein. Proc Natl Acad Sci U S A, 94, 6623-6628. 73. Masuda, T., Ito, Y., Terada, T., Shibata, T. and Mikawa, T. (2009) Anon-canonical DNA structure enables homologous recombination in various genetic systems. J Biol Chem, 284, 30230-30239. | |
| 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.subject | 同源重組蛋白酶 | zh_TW |
| dc.subject | 同源重組 | zh_TW |
| dc.subject | 縮胺酸 | zh_TW |
| dc.subject | 去氧核醣核酸 | zh_TW |
| dc.subject | 左旋絲狀體 | zh_TW |
| dc.subject | X 光繞射 | zh_TW |
| dc.subject | 結構 | zh_TW |
| dc.subject | RecA | en |
| dc.subject | drug design and strand exchange | en |
| dc.subject | left-handed filaments | en |
| dc.subject | small peptides | en |
| dc.subject | DNA repair | en |
| dc.subject | homologous recombination | 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-99-1.pdf 未授權公開取用 | 8.11 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。