臺灣博碩士論文加值系統

拓樸異構酶普遍存在於所有細胞，負責解決各種因DNA代謝過程，例如：複製、轉錄、染色體分離、……等，所造成DNA結構的拓樸學問題。近年來發現人類細胞有兩種拓樸酶III，分別被稱為hTOP3α和hTOP3β，愈來愈多的證據說明，這兩種同功酶可能在細胞中執行不同的功能，為了進一步探討同功酶的特異性，我們首先使用桿狀病毒與細菌表現系統來大量表達hTOP3α和hTOP3β，然後進行蛋白質純化。純化出的酵素在試管中測試其生化特性，結果發現採用不同受質DNA明顯影響了重組hTOP3α和hTOP3β的活性表現：兩者對一般帶負超螺旋的質體DNA僅具微弱解螺旋能力；但兩種同功酶都能順利將超負超螺旋受質解開成負超螺旋。上述觀察與前人對果蠅Top3的實驗結論相符。簡言之，本篇論文成功大量表達了hTOP3α和hTOP3β同功酶，並且建立了一個快速、容易且有效的純化方式，純化出的酵素將可被應用於未來更多的研究。
此外，之前已有報導證明在大腸桿菌中，過量表達Tnp將會抑制TopA；於酵母菌的研究亦顯示，過量表達Tnp也會抑制ALT端粒延長機制。綜合以上兩項實驗結果，以及TopA與hTOP3結構上的高度相似性，還有TOP3在ALT路徑扮演之角色，我們提出Tnp會抑制hTOP3的假設。為證實此假設，我們建構帶有Tnp、Inh、55aa的質體，並將之送入酵母菌或哺乳動物細胞以利進一步的研究。

Topoisomerases are ubiquitous enzymes found in almost all cells and play important roles in many biological processes, such as DNA replication, transcription, and chromosome condensation by regulating the topology of DNA. Two isoforms of topoisomerase III, hTOP3alpha and hTOP3beta, have been recently identified in human cells. Accumulating evidences indicated that hTOP3alpha and hTOP3beta might exhibit distinct cellular functions. In order to further clarify isozyme-specific activities, we first employed both baculoviral and bacterial expression systems to produce large amounts of enzymes for purification. The biochemical properties of purified enzymes were then tested in vitro. using negative supercoiled or hyper-negative supercoiled substrates. Despite of a rather weak relaxation activity against usual plasmid DNA, purifie hTOP3alpha and hTOP3beta showed their preferences for hyper-negative supercoiled structure which might have more single-stranded regions exposed. This result is in consistent with experiments done with Drosophila Top3. Taken together, we successfully overexpressed both hTOP3 isozymes and also developed a fast, easy, and efficient purification protocol. More studies could be done with these purified products in the future.
It has been proved that overexpression of Tnp in E. coli would result in titration of TopA. The previous research done in S. cerevisiae also led to the conclusion that Tnp could inhibit ALT pathway. Because of the structure similarity between TopA and hTOP3, and the role Top3 played in ALT pathway, the hypothesis that Tnp might work through inhibiting Top3 was arised. We then constructed plasmids encoding Tnp, 55aa, and Inh. The recombinant plasmid will be sent into yeast or mammalian cells for further examination.

目錄……………………………………………………………………3
英文摘要………………………………………………………………4
中文摘要………………………………………………………………5
緒論……………………………………………………………………6
實驗目標………………………………………………………………19
材料與方法……………………………………………………………20
結果……………………………………………………………………32
討論……………………………………………………………………42
圖表……………………………………………………………………46
參考資料………………………………………………………………64

Abraham, R.T. (2001) Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev, 15, 2177-2196.
Aoki, K., Ishida, R. and Kasai, M. (1997) Isolation and characterization of a cDNA encoding a Translin-like protein, TRAX. FEBS Lett, 401, 109-112.
Bachrati, C.Z. and Hickson, I.D. (2003) RecQ helicases: suppressors of tumorigenesis and premature aging. Biochem J, 374, 577-606.
Bailis, A.M., Arthur, L. and Rothstein, R. (1992) Genome rearrangement in top3 mutants of Saccharomyces cerevisiae requires a functional RAD1 excision repair gene. Mol Cell Biol, 12, 4988-4993.
Bennett, R.J., Noirot-Gros, M.F. and Wang, J.C. (2000) Interaction between yeast sgs1 helicase and DNA topoisomerase III. J Biol Chem, 275, 26898-26905.
Brachmann, R.K. and Boeke, J.D. (1997) Tag games in yeast: the two-hybrid system and beyond. Curr Opin Biotechnol, 8, 561-568.
Chakraverty, R.K., Kearsey, J.M., Oakley, T.J., Grenon, M., de La Torre Ruiz, M.A., Lowndes, N.F. and Hickson, I.D. (2001) Topoisomerase III acts upstream of Rad53p in the S-phase DNA damage checkpoint. Mol Cell Biol, 21, 7150-7162.
Champoux, J.J. (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem, 70, 369-413.
DiGate, R.J. and Marians, K.J. (1992) Escherichia coli topoisomerase III-catalyzed cleavage of RNA. J Biol Chem, 267, 20532-20535.
Drolet, M., Bi, X. and Liu, L.F. (1994) Hypernegative supercoiling of the DNA template during transcription elongation in vitro. J Biol Chem, 269, 2068-2074.
Drolet, M., Phoenix, P., Menzel, R., Masse, E., Liu, L.F. and Crouch, R.J. (1995) Overexpression of RNase H partially complements the growth defect of an Escherichia coli delta topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase I. Proc Natl Acad Sci U S A, 92, 3526-3530.
Dulbecco, R. and Vogt, M. (1963) Evidence For A Ring Structure Of Polyoma Virus Dna. Proc Natl Acad Sci U S A, 50, 236-243.
Dutertre, S., Ababou, M., Onclercq, R., Delic, J., Chatton, B., Jaulin, C. and Amor-Gueret, M. (2000) Cell cycle regulation of the endogenous wild type Bloom''s syndrome DNA helicase. Oncogene, 19, 2731-2738.
Ellis, N.A., Groden, J., Ye, T.Z., Straughen, J., Lennon, D.J., Ciocci, S., Proytcheva, M. and German, J. (1995) The Bloom''s syndrome gene product is homologous to RecQ helicases. Cell, 83, 655-666.
Fabre, F., Chan, A., Heyer, W.D. and Gangloff, S. (2002) Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication. Proc Natl Acad Sci U S A, 99, 16887-16892.
Frei, C. and Gasser, S.M. (2000) The yeast Sgs1p helicase acts upstream of Rad53p in the DNA replication checkpoint and colocalizes with Rad53p in S-phase-specific foci. Genes Dev, 14, 81-96.
Fricke, W.M., Kaliraman, V. and Brill, S.J. (2001) Mapping the DNA topoisomerase III binding domain of the Sgs1 DNA helicase. J Biol Chem, 276, 8848-8855.
Fritz, E., Elsea, S.H., Patel, P.I. and Meyn, M.S. (1997) Overexpression of a truncated human topoisomerase III partially corrects multiple aspects of the ataxia-telangiectasia phenotype. Proc Natl Acad Sci U S A, 94, 4538-4542.
Gangloff, S., McDonald, J.P., Bendixen, C., Arthur, L. and Rothstein, R. (1994) The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase. Mol Cell Biol, 14, 8391-8398.
Goodwin, A., Wang, S.W., Toda, T., Norbury, C. and Hickson, I.D. (1999) Topoisomerase III is essential for accurate nuclear division in Schizosaccharomyces pombe. Nucleic Acids Res, 27, 4050-4058.
Hanai, R., Caron, P.R. and Wang, J.C. (1996) Human TOP3: a single-copy gene encoding DNA topoisomerase III. Proc Natl Acad Sci U S A, 93, 3653-3657.
Harmon, F.G., Brockman, J.P. and Kowalczykowski, S.C. (2003) RecQ helicase stimulates both DNA catenation and changes in DNA topology by topoisomerase III. J Biol Chem, 278, 42668-42678.
Harmon, F.G., DiGate, R.J. and Kowalczykowski, S.C. (1999) RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination. Mol Cell, 3, 611-620.
Hiasa, H., DiGate, R.J. and Marians, K.J. (1994) Decatenating activity of Escherichia coli DNA gyrase and topoisomerases I and III during oriC and pBR322 DNA replication in vitro. J Biol Chem, 269, 2093-2099.
Hickson, I.D. (2003) RecQ helicases: caretakers of the genome. Nat Rev Cancer, 3, 169-178.
Hsiang, Y.H., Wu, H.Y. and Liu, L.F. (1988) Proliferation-dependent regulation of DNA topoisomerase II in cultured human cells. Cancer Res, 48, 3230-3235.
James, P., Halladay, J. and Craig, E.A. (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics, 144, 1425-1436.
Kaliraman, V., Mullen, J.R., Fricke, W.M., Bastin-Shanower, S.A. and Brill, S.J. (2001) Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease. Genes Dev, 15, 2730-2740.
Kim, R.A., Caron, P.R. and Wang, J.C. (1995) Effects of yeast DNA topoisomerase III on telomere structure. Proc Natl Acad Sci U S A, 92, 2667-2671.
Kim, R.A. and Wang, J.C. (1992) Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase. J Biol Chem, 267, 17178-17185.
Kitao, S., Shimamoto, A., Goto, M., Miller, R.W., Smithson, W.A., Lindor, N.M. and Furuichi, Y. (1999) Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome. Nat Genet, 22, 82-84.
Kobayashi, M. and Hanai, R. (2001) M phase-specific association of human topoisomerase IIIbeta with chromosomes. Biochem Biophys Res Commun, 287, 282-287.
Kwan, K.Y., Moens, P.B. and Wang, J.C. (2003) Infertility and aneuploidy in mice lacking a type IA DNA topoisomerase III beta. Proc Natl Acad Sci U S A, 100, 2526-2531.
Kwan, K.Y. and Wang, J.C. (2001) Mice lacking DNA topoisomerase IIIbeta develop to maturity but show a reduced mean lifespan. Proc Natl Acad Sci U S A, 98, 5717-5721.
Laursen, L.V., Ampatzidou, E., Andersen, A.H. and Murray, J.M. (2003) Role for the fission yeast RecQ helicase in DNA repair in G2. Mol Cell Biol, 23, 3692-3705.
Li, T.K. and Liu, L.F. (2001) Tumor cell death induced by topoisomerase-targeting drugs. Annu Rev Pharmacol Toxicol, 41, 53-77.
Li, W., Kim, S.M., Lee, J. and Dunphy, W.G. (2004) Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases. J Cell Biol, 165, 801-812.
Li, W. and Wang, J.C. (1998) Mammalian DNA topoisomerase IIIalpha is essential in early embryogenesis. Proc Natl Acad Sci U S A, 95, 1010-1013.
Liu, L.F. and Wang, J.C. (1987) Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A, 84, 7024-7027.
Liu, Q., Guntuku, S., Cui, X.S., Matsuoka, S., Cortez, D., Tamai, K., Luo, G., Carattini-Rivera, S., DeMayo, F., Bradley, A., Donehower, L.A. and Elledge, S.J. (2000) Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev, 14, 1448-1459.
Maftahi, M., Han, C.S., Langston, L.D., Hope, J.C., Zigouras, N. and Freyer, G.A. (1999) The top3(+) gene is essential in Schizosaccharomyces pombe and the lethality associated with its loss is caused by Rad12 helicase activity. Nucleic Acids Res, 27, 4715-4724.
Matsuoka, S., Huang, M. and Elledge, S.J. (1998) Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science, 282, 1893-1897.
Matsuoka, S., Rotman, G., Ogawa, A., Shiloh, Y., Tamai, K. and Elledge, S.J. (2000) Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc Natl Acad Sci U S A, 97, 10389-10394.
Meetei, A.R., Sechi, S., Wallisch, M., Yang, D., Young, M.K., Joenje, H., Hoatlin, M.E. and Wang, W. (2003) A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome. Mol Cell Biol, 23, 3417-3426.
Miyajima, A., Seki, M., Onoda, F., Shiratori, M., Odagiri, N., Ohta, K., Kikuchi, Y., Ohno, Y. and Enomoto, T. (2000) Sgs1 helicase activity is required for mitotic but apparently not for meiotic functions. Mol Cell Biol, 20, 6399-6409.
Mondragon, A. and DiGate, R. (1999) The structure of Escherichia coli DNA topoisomerase III. Structure Fold Des, 7, 1373-1383.
Mullen, J.R., Kaliraman, V. and Brill, S.J. (2000) Bipartite structure of the SGS1 DNA helicase in Saccharomyces cerevisiae. Genetics, 154, 1101-1114.
Ng, S.W., Liu, Y., Hasselblatt, K.T., Mok, S.C. and Berkowitz, R.S. (1999) A new human topoisomerase III that interacts with SGS1 protein. Nucleic Acids Res, 27, 993-1000.
Oh, M., Choi, I.S. and Park, S.D. (2002) Topoisomerase III is required for accurate DNA replication and chromosome segregation in Schizosaccharomyces pombe. Nucleic Acids Res, 30, 4022-4031.
Painter, R.B. and Young, B.R. (1980) Radiosensitivity in ataxia-telangiectasia: a new explanation. Proc Natl Acad Sci U S A, 77, 7315-7317.
Plank, J.L., Chu, S.H., Pohlhaus, J.R., Wilson-Sali, T. and Hsieh, T.S. (2005) Drosophila melanogaster topoisomerase IIIalpha preferentially relaxes a positively or negatively supercoiled bubble substrate and is essential during development. J Biol Chem, 280, 3564-3573.
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.
Sherr, C.J. (2004) Principles of tumor suppression. Cell, 116, 235-246.
Shimamoto, A., Nishikawa, K., Kitao, S. and Furuichi, Y. (2000) Human RecQ5beta, a large isomer of RecQ5 DNA helicase, localizes in the nucleoplasm and interacts with topoisomerases 3alpha and 3beta. Nucleic Acids Res, 28, 1647-1655.
Shor, E., Gangloff, S., Wagner, M., Weinstein, J., Price, G. and Rothstein, R. (2002) Mutations in homologous recombination genes rescue top3 slow growth in Saccharomyces cerevisiae. Genetics, 162, 647-662.
Spellman, P.T., Sherlock, G., Zhang, M.Q., Iyer, V.R., Anders, K., Eisen, M.B., Brown, P.O., Botstein, D. and Futcher, B. (1998) Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol Biol Cell, 9, 3273-3297.
Suzuki, Y., Demoliere, C., Kitamura, D., Takeshita, H., Deuschle, U. and Watanabe, T. (1997) HAX-1, a novel intracellular protein, localized on mitochondria, directly associates with HS1, a substrate of Src family tyrosine kinases. J Immunol, 158, 2736-2744.
Tsai, HJ, and Teng, SC. (2004) DNA topoisomerase III involves in telomere-telomere recombination pathway. Institute of Microbiology School of Medicine National Taiwan University Master Thesis
Vinograd, J., Lebowitz, J., Radloff, R., Watson, R. and Laipis, P. (1965) The twisted circular form of polyoma viral DNA. Proc Natl Acad Sci U S A, 53, 1104-1111.
Wallis, J.W., Chrebet, G., Brodsky, G., Rolfe, M. and Rothstein, R. (1989) A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell, 58, 409-419.
Wang, J.C. (1971) Interaction between DNA and an Escherichia coli protein omega. J Mol Biol, 55, 523-533.
Wang, J.C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol, 3, 430-440.
Wang, T.F. and Kung, W.M. (2002) Supercomplex formation between Mlh1-Mlh3 and Sgs1-Top3 heterocomplexes in meiotic yeast cells. Biochem Biophys Res Commun, 296, 949-953.
Wang, Y., Cortez, D., Yazdi, P., Neff, N., Elledge, S.J. and Qin, J. (2000) BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev, 14, 927-939.
Watson, J.D. and Crick, F.H. (1953) Genetical implications of the structure of deoxyribonucleic acid. Nature, 171, 964-967.
Watson, J.D. and Crick, F.H. (1953) Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature, 171, 737-738.
Watt, P.M., Hickson, I.D., Borts, R.H. and Louis, E.J. (1996) SGS1, a homologue of the Bloom''s and Werner''s syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae. Genetics, 144, 935-945.
Watt, P.M., Louis, E.J., Borts, R.H. and Hickson, I.D. (1995) Sgs1: a eukaryotic homolog of E. coli RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation. Cell, 81, 253-260.
Weil, R. and Vinograd, J. (1963) The Cyclic Helix And Cyclic Coil Forms Of Polyoma Viral Dna. Proc Natl Acad Sci U S A, 50, 730-738.
Weinert, T.A. and Hartwell, L.H. (1988) The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cerevisiae. Science, 241, 317-322.
Wilson-Sali, T. and Hsieh, T.S. (2002) Generation of double-stranded breaks in hypernegatively supercoiled DNA by Drosophila topoisomerase IIIbeta, a type IA enzyme. J Biol Chem, 277, 26865-26871.
Wilson-Sali, T. and Hsieh, T.S. (2002) Preferential cleavage of plasmid-based R-loops and D-loops by Drosophila topoisomerase IIIbeta. Proc Natl Acad Sci U S A, 99, 7974-7979.
Wilson, T.M., Chen, A.D. and Hsieh, T. (2000) Cloning and characterization of Drosophila topoisomerase IIIbeta. Relaxation of hypernegatively supercoiled DNA. J Biol Chem, 275, 1533-1540.
Woessner, R.D., Mattern, M.R., Mirabelli, C.K., Johnson, R.K. and Drake, F.H. (1991) Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH-3T3 cells. Cell Growth Differ, 2, 209-214.
Wu, L., Davies, S.L., Levitt, N.C. and Hickson, I.D. (2001) Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51. J Biol Chem, 276, 19375-19381.
Wu, L., Davies, S.L., North, P.S., Goulaouic, H., Riou, J.F., Turley, H., Gatter, K.C. and Hickson, I.D. (2000) The Bloom''s syndrome gene product interacts with topoisomerase III. J Biol Chem, 275, 9636-9644.
Yigit, H. and Reznikoff, W.S. (1998) Escherichia coli DNA topoisomerase I and suppression of killing by Tn5 transposase overproduction: topoisomerase I modulates Tn5 transposition. J Bacteriol, 180, 5866-5874.
Yigit, H. and Reznikoff, W.S. (1999) Escherichia coli DNA topoisomerase I copurifies with Tn5 transposase, and Tn5 transposase inhibits topoisomerase I. J Bacteriol, 181, 3185-3192.
Yu, C.E., Oshima, J., Fu, Y.H., Wijsman, E.M., Hisama, F., Alisch, R., Matthews, S., Nakura, J., Miki, T., Ouais, S., Martin, G.M., Mulligan, J. and Schellenberg, G.D. (1996) Positional cloning of the Werner''s syndrome gene. Science, 272, 258-262.
Zhao, H. and Piwnica-Worms, H. (2001) ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol, 21, 4129-4139.