請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67035完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林敬哲(Jing-Jer Lin) | |
| dc.contributor.author | Yi-Yun Lin | en |
| dc.contributor.author | 林易運 | zh_TW |
| dc.date.accessioned | 2021-06-17T01:17:57Z | - |
| dc.date.available | 2022-09-08 | |
| dc.date.copyright | 2017-09-08 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-14 | |
| dc.identifier.citation | Baumann, P., and Cech, T.R. (2001). Pot1, the putative telomere end-binding protein in fission yeast and humans. Science 292, 1171-1175.
Bilaud, T., Brun, C., Ancelin, K., Koering, C.E., Laroche, T., and Gilson, E. (1997). Telomeric localization of TRF2, a novel human telobox protein. Nat Genet 17, 236-239. Blackburn, E.H. (1990). Telomeres: structure and synthesis. J Biol Chem 265, 5919-5921. Bosoy, D., and Lue, N.F. (2004). Yeast telomerase is capable of limited repeat addition processivity. Nucleic Acids Res 32, 93-101. Chandra, A., Hughes, T.R., Nugent, C.I., and Lundblad, V. (2001). Cdc13 both positively and negatively regulates telomere replication. Genes Dev 15, 404-414. Chen, J., Le, S., Basu, A., Chazin, W.J., and Yan, J. (2015). Mechanochemical regulations of RPA's binding to ssDNA. Sci Rep 5, 9296. Chen, L.Y., Redon, S., and Lingner, J. (2012). The human CST complex is a terminator of telomerase activity. Nature 488, 540-544. Chen, Y.F., Lu, C.Y., Lin, Y.C., Yu, T.Y., Chang, C.P., Li, J.R., Li, H.W., and Lin, J.J. (2016). Modulation of yeast telomerase activity by Cdc13 and Est1 in vitro. Sci Rep 6, 34104. Counter, C.M., Meyerson, M., Eaton, E.N., and Weinberg, R.A. (1997). The catalytic subunit of yeast telomerase. Proc Natl Acad Sci U S A 94, 9202-9207. Dandjinou, A.T., Levesque, N., Larose, S., Lucier, J.F., Abou Elela, S., and Wellinger, R.J. (2004). 'A phylogenetically based secondary structure for the yeast telomerase RNA'. Curr Biol 14, p. 1148-1158. de Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 19, 2100-2110. DeZwaan, D.C., and Freeman, B.C. (2009). The conserved Est1 protein stimulates telomerase DNA extension activity. Proc Natl Acad Sci U S A 106, 17337-17342. Evans, S.K., and Lundblad, V. (1999). Est1 and Cdc13 as comediators of telomerase access. Science 286, 117-120. Evans, S.K., and Lundblad, V. (2002). The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance. Genetics 162, 1101-1115. Fairman, M.P., and Stillman, B. (1988). Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J 7, 1211-1218. Gao, H., Cervantes, R.B., Mandell, E.K., Otero, J.H., and Lundblad, V. (2007). RPA-like proteins mediate yeast telomere function. Nat Struct Mol Biol 14, 208-214. Garvik, B., Carson, M., and Hartwell, L. (1995). Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint. Mol Cell Biol 15, 6128-6138. Gelinas, A.D., Paschini, M., Reyes, F.E., Heroux, A., Batey, R.T., Lundblad, V., and Wuttke, D.S. (2009). Telomere capping proteins are structurally related to RPA with an additional telomere-specific domain. Proc Natl Acad Sci U S A 106, 19298-19303. Giraud-Panis, M.J., Teixeira, M.T., Geli, V., and Gilson, E. (2010). CST meets shelterin to keep telomeres in check. Mol Cell 39, 665-676. Gottschling, D.E., Aparicio, O.M., Billington, B.L., and Zakian, V.A. (1990). Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63, 751-762. Grandin, N., Damon, C., and Charbonneau, M. (2000). Cdc13 cooperates with the yeast Ku proteins and Stn1 to regulate telomerase recruitment. Mol Cell Biol 20, 8397-8408. Grandin, N., Damon, C., and Charbonneau, M. (2001). Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13. EMBO J 20, 1173-1183. Grandin, N., Reed, S.I., and Charbonneau, M. (1997). Stn1, a new Saccharomyces cerevisiae protein, is implicated in telomere size regulation in association with Cdc13. Genes Dev 11, 512-527. Greider, C.W., and Blackburn, E.H. (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43, 405-413. Griffith, J.D., Comeau, L., Rosenfield, S., Stansel, R.M., Bianchi, A., Moss, H., and de Lange, T. (1999). Mammalian telomeres end in a large duplex loop. Cell 97, 503-514. Hang, L.E., Liu, X., Cheung, I., Yang, Y., and Zhao, X. (2011). SUMOylation regulates telomere length homeostasis by targeting Cdc13. Nat Struct Mol Biol 18, 920-926. Houghtaling, B.R., Cuttonaro, L., Chang, W., and Smith, S. (2004). A dynamic molecular link between the telomere length regulator TRF1 and the chromosome end protector TRF2. Curr Biol 14, 1621-1631. Hsu, C.L., Chen, Y.S., Tsai, S.Y., Tu, P.J., Wang, M.J., and Lin, J.J. (2004). Interaction of Saccharomyces Cdc13p with Pol1p, Imp4p, Sir4p and Zds2p is involved in telomere replication, telomere maintenance and cell growth control. Nucleic Acids Res 32, 511-521. Hughes, T.R., Weilbaecher, R.G., Walterscheid, M., and Lundblad, V. (2000). Identification of the single-strand telomeric DNA binding domain of the Saccharomyces cerevisiae Cdc13 protein. Proc Natl Acad Sci U S A 97, 6457-6462. Kastan, M.B., and Bartek, J. (2004). Cell-cycle checkpoints and cancer. Nature 432, 316-323. Kim, C., Paulus, B.F., and Wold, M.S. (1994). Interactions of human replication protein A with oligonucleotides. Biochemistry 33, 14197-14206. Lebo, K.J., Niederer, R.O., and Zappulla, D.C. (2015). A second essential function of the Est1-binding arm of yeast telomerase RNA. RNA 21, 862-876. Lendvay, T.S., Morris, D.K., Sah, J., Balasubramanian, B., and Lundblad, V. (1996). Senescence mutants of Saccharomyces cerevisiae with a defect in telomere replication identify three additional EST genes. Genetics 144, 1399-1412. Lin, J.J., and Zakian, V.A. (1996). The Saccharomyces CDC13 protein is a single-strand TG1-3 telomeric DNA-binding protein in vitro that affects telomere behavior in vivo. Proc Natl Acad Sci U S A 93, 13760-13765. Liu, D., O'Connor, M.S., Qin, J., and Songyang, Z. (2004). Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins. J Biol Chem 279, 51338-51342. Lundblad, V., and Szostak, J.W. (1989). A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 57, 633-643. Lustig, A.J. (2001). Cdc13 subcomplexes regulate multiple telomere functions. Nat Struct Biol 8, 297-299. Marcand, S., Brevet, V., Mann, C., and Gilson, E. (2000). Cell cycle restriction of telomere elongation. Curr Biol 10, 487-490. Mason, M., Wanat, J.J., Harper, S., Schultz, D.C., Speicher, D.W., Johnson, F.B., and Skordalakes, E. (2013). Cdc13 OB2 dimerization required for productive Stn1 binding and efficient telomere maintenance. Structure 21, 109-120. Mitchell, M.T., Smith, J.S., Mason, M., Harper, S., Speicher, D.W., Johnson, F.B., and Skordalakes, E. (2010). Cdc13 N-terminal dimerization, DNA binding, and telomere length regulation. Mol Cell Biol 30, 5325-5334. Miyake, Y., Nakamura, M., Nabetani, A., Shimamura, S., Tamura, M., Yonehara, S., Saito, M., and Ishikawa, F. (2009). RPA-like mammalian Ctc1-Stn1-Ten1 complex binds to single-stranded DNA and protects telomeres independently of the Pot1 pathway. Mol Cell 36, 193-206. Mozdy, A.D., and Cech, T.R. (2006). Low abundance of telomerase in yeast: implications for telomerase haploinsufficiency. RNA 12, p. 1721-1737. Nugent, C.I., Hughes, T.R., Lue, N.F., and Lundblad, V. (1996). Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance. Science 274, 249-252. Olovnikov, A.M. (1973). A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol 41, 181-190. Pennock, E., Buckley, K., and Lundblad, V. (2001). Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell 104, 387-396. Pfeiffer, V., and Lingner, J. (2013). Replication of telomeres and the regulation of telomerase. Cold Spring Harb Perspect Biol 5, a010405. Price, C.M., Boltz, K.A., Chaiken, M.F., Stewart, J.A., Beilstein, M.A., and Shippen, D.E. (2010). Evolution of CST function in telomere maintenance. Cell Cycle 9, 3157-3165. Puglisi, A., Bianchi, A., Lemmens, L., Damay, P., and Shore, D. (2008). Distinct roles for yeast Stn1 in telomere capping and telomerase inhibition. EMBO J 27, 2328-2339. Qi, H., and Zakian, V.A. (2000). The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase alpha and the telomerase-associated est1 protein. Genes Dev 14, 1777-1788. Sancar, A., Lindsey-Boltz, L.A., Unsal-Kacmaz, K., and Linn, S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73, 39-85. Sandell, L.L., and Zakian, V.A. (1993). Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75, 729-739. Seto, A.G., Livengood, A.J., Tzfati, Y., Blackburn, E.H., and Cech, T.R. (2002). A bulged stem tethers Est1p to telomerase RNA in budding yeast. Genes Dev 16, 2800-2812. Shampay, J., Szostak, J.W., and Blackburn, E.H. (1984). DNA sequences of telomeres maintained in yeast. Nature 310, 154-157. Singer, M.S., and Gottschling, D.E. (1994). TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science 266, 404-409. Smith, J.R., and Pereira-Smith, O.M. (1996). Replicative senescence: implications for in vivo aging and tumor suppression. Science 273, 63-67. Stansel, R.M., de Lange, T., and Griffith, J.D. (2001). T-loop assembly in vitro involves binding of TRF2 near the 3' telomeric overhang. EMBO J 20, 5532-5540. Steinberg-Neifach, O., and Lue, N.F. (2015). Telomere DNA recognition in Saccharomycotina yeast: potential lessons for the co-evolution of ssDNA and dsDNA-binding proteins and their target sites. Front Genet 6, 162. Sun, J., Yu, E.Y., Yang, Y., Confer, L.A., Sun, S.H., Wan, K., Lue, N.F., and Lei, M. (2009). Stn1-Ten1 is an Rpa2-Rpa3-like complex at telomeres. Genes Dev 23, 2900-2914. Takeyasu, K., Omote, H., Nettikadan, S., Tokumasu, F., Iwamoto-Kihara, A., and Futai, M. (1996). Molecular imaging of Escherichia coli F0F1-ATPase in reconstituted membranes using atomic force microscopy. FEBS Lett 392, 110-113. Teixeira, M.T., Arneric, M., Sperisen, P., and Lingner, J. (2004). Telomere length homeostasis is achieved via a switch between telomerase- extendible and -nonextendible states. Cell 117, 323-335. Tseng, S.F., Lin, J.J., and Teng, S.C. (2006). The telomerase-recruitment domain of the telomere binding protein Cdc13 is regulated by Mec1p/Tel1p-dependent phosphorylation. Nucleic Acids Res 34, 6327-6336. Tucey, T.M., and Lundblad, V. (2013). A yeast telomerase complex containing the Est1 recruitment protein is assembled early in the cell cycle. Biochemistry 52, 1131-1133. Virta-Pearlman, V., Morris, D.K., and Lundblad, V. (1996). Est1 has the properties of a single-stranded telomere end-binding protein. Genes Dev 10, 3094-3104. Waga, S., and Stillman, B. (1998). The DNA replication fork in eukaryotic cells. Annu Rev Biochem 67, 721-751. Wobbe, C.R., Weissbach, L., Borowiec, J.A., Dean, F.B., Murakami, Y., Bullock, P., and Hurwitz, J. (1987). Replication of simian virus 40 origin-containing DNA in vitro with purified proteins. Proc Natl Acad Sci U S A 84, 1834-1838. Wold, M.S. (1997). Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu Rev Biochem 66, 61-92. Wold, M.S., and Kelly, T. (1988). Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A 85, 2523-2527. Wu, Y., and Zakian, V.A. (2011). The telomeric Cdc13 protein interacts directly with the telomerase subunit Est1 to bring it to telomeric DNA ends in vitro. Proc Natl Acad Sci U S A 108, 20362-20369. Zakian, V.A. (1995). Telomeres: beginning to understand the end. Science 270, 1601-1607. Zakian, V.A. (1996). Structure, function, and replication of Saccharomyces cerevisiae telomeres. Annu Rev Genet 30, 141-172. Zappulla, D.C., and Cech, T.R. (2004). Yeast telomerase RNA: a flexible scaffold for protein subunits. Proc Natl Acad Sci U S A 101, 10024-10029. Zappulla, D.C., Roberts, J.N., Goodrich, K.J., Cech, T.R., and Wuttke, D.S. (2009). Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p. Nucleic Acids Res 37, 354-367. Zhong, Z., Shiue, L., Kaplan, S., and de Lange, T. (1992). A mammalian factor that binds telomeric TTAGGG repeats in vitro. Mol Cell Biol 12, 4834-4843. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67035 | - |
| dc.description.abstract | 酵母菌端粒酶的活性會受到端粒相關蛋白的調控，而Cdc13 在其中扮演重要的角色。Cdc13為單股端粒結合蛋白，其可將端粒酶藉由與另外一個調控蛋白Est1結合而補充至端粒末端以促使端粒的合成。Cdc13也會跟Stn1及Ten1結合形成複合物以抑制端粒酶的活性。過去已被報導Cdc13本身有抑制端粒酶的活性，而我們發現Stn1可以加強Cdc13抑制端粒酶活性的效果。為了瞭解這些蛋白是如何調控端粒的長度，我們藉由建立單分子栓球的實驗以應用於檢測這些蛋白和DNA之間的作用關係。我們藉由將Cdc13接上栓球以觀察Cdc13如何與DNA作用的實驗發現布朗運動的情況會受到抑制。為了瞭解Cdc13是如何去造成DNA布朗運動變化，我們運用追蹤布朗運動隨時間變化的路徑觀察DNA接上栓球後布朗運動是否受到Cdc13影響，發現Cdc13會造成DNA的長度被濃縮，並且變化的模式可以主要分成三種：一階變化，二階變化，及消長型變化。藉由觀察布朗運動變化所需的時間，我們發現Cdc13和DNA的作用過程是一種動態變化，並且每次變化的布朗運動值是以接近8的倍數來變化．除此之外，我們藉由原子力學顯微鏡的實驗更進一步的觀察Cdc13是如何造成DNA有長度上的變化，我們發現當DNA接上蛋白時，DNA的長度會明顯縮短，結果和單分子拴球實驗有很高的共同性，同時我們分析了每次DNA縮短的值也接近於用單分子拴球所得到的數值，藉由這些單分子的實驗我們發現Cdc13具備改變端粒構型的能力，這可能說明了Cdc13調控端粒的方式就是藉由改變端粒的構型以保護端粒不被其他酵素攻擊。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-17T01:17:57Z (GMT). No. of bitstreams: 1 ntu-106-R04442007-1.pdf: 2913730 bytes, checksum: 02c59aa52cbc05ec304ad84b31399782 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | Table of Contents
謝辭 ii 摘要 v Abstract vi Introduction 1 Materials and Methods 8 1. Strain 8 2. Oligonucleotide (5’→3’) 8 3. Media and plates 9 4. Purification of Est2/TLC1 RNP from yeast 11 5. Purification of 6xHis-Cdc13 from insect cell 12 6. Purification of 6xHis-Smt3-Stn1 16 7. Measurement of the protein concentration 17 8. SDS- Poly-acrylamide-gel-electrophoresis (SDS-PAGE) 17 9. Preparation of isotope-labeled oligonucleotides 18 10. Electrophoretic mobility shift assay 19 11. Primer extension assay 19 12. Tethered particle motion (TPM) 21 13. Atomic force microscope (AFM) 27 Results 31 1. Purification of recombinant proteins and activity test 31 I. Est2/Tlc1 RNP purification and telomerase activity assay 31 II. 6x-His Cdc13 purification and single strand telomere binding ability test 31 III. Purification of Stn1 and single-strand telomere binding ability test 32 2. Stn1 enhances Cdc13 inhibitory effect on telomerase activity 32 3. Cdc13-beads binding on DNA causes BM shrinkage 33 4. Cdc13-beads prefer to bind telomere substrate. 35 5. Cdc13 condenses DNA length to cause BM shrinkage 37 6. Cdc13 condenses DNA dynamically and presents more severe decrease level on longer substrates 39 7. Cdc13 wrapping DNA visualized under AFM 40 Discussion 43 Reference 49 List of Table 56 List of Figure 64 Appendix 84 Figure 1. Primer extension assay to test telomerase activity 64 Figure 2. Purification of 6xHis-Cdc13 and gel shift assay 65 Figure 3. Purification of 6xHis-Smt3-Stn1 and gel shift assay 66 Figure 4. Stn1 enhances Cdc13 inhibitory effect on telomerase 67 Figure 5. Single-molecule TPM experiment 68 Figure 6. Illustration of preparation of 3’-overhang telomere substrate for single- molecule approach 69 Figure 7. Cdc13 decreases DNA BM by multiple-run reaction and depends on dose 70 Figure 8. Cdc13 decreases DNA BM by multiple-run reaction 71 Figure 9. Cdc13 prefers to bind telomere substrate 72 Figure 10. Cdc13-beads does not affect the binding property of Cdc13 73 Figure 11. Brownian motion histogram of TPM substrates 74 Figure 13. Analysis the characteristic of the BM decrease population when Cdc13 incubate with telomere 77 Figure 14. Analysis of BM distribution when BM decrease and the differences for BM change 78 Figure 15. Representative AFM images of the analyzed complexes 79 Figure 16. AFM measurement of molecules parameters 80 Figure 17. 2D-dot plot for analysis the AFM measurement of molecules parameters 82 Figure 18. The model of Cdc13-Telomere complex structure alteration 83 | |
| dc.language.iso | en | |
| dc.subject | 端粒? | zh_TW |
| dc.subject | 端粒 | zh_TW |
| dc.subject | 端粒結合蛋白 | zh_TW |
| dc.subject | 單分子實驗 | zh_TW |
| dc.subject | 老化 | zh_TW |
| dc.subject | Senescence | en |
| dc.subject | Telomerase | en |
| dc.subject | Telomere | en |
| dc.subject | Telomere-binding protein | en |
| dc.subject | Single-molecule experiment | en |
| dc.title | 利用單分子技術去探討端粒結合蛋白對於端粒酶的調控機制 | zh_TW |
| dc.title | Applying Single-Molecule Approaches to Investigate the Regulatory Mechanism of Telomere Binding Proteins on Telomerase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李弘文,大庭良介 | |
| dc.subject.keyword | 端粒,端粒?,老化,單分子實驗,端粒結合蛋白, | zh_TW |
| dc.subject.keyword | Telomere,Telomerase,Senescence,Single-molecule experiment,Telomere-binding protein, | en |
| dc.relation.page | 88 | |
| dc.identifier.doi | 10.6342/NTU201703125 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-14 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-106-1.pdf 未授權公開取用 | 2.85 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。