臺灣博碩士論文加值系統

乳酸菌 Lactobacillus casei 中所分離出來的 ISLC3（AF445084） 為轉位子 IS3 family 成員之一，長1,351 bp，可轉譯 382 個胺基酸。ISLC3 包含兩個開放編譯框 (open reading frame)。IS3 family 見存於在革蘭氏陽性菌 G（+）與革蘭氏陰性菌 G（-）中約40種細菌，並以非複製形態進行轉位機制。轉位子是很好的基因突變體，轉位子與基因體 DNA 重組或質體嵌入基因體息息相關，具有重塑基因庫的能力，在基因遺傳和演化上，扮演極為重要的角色。但是太頻繁的轉位作用所伴隨產生的基因組重組現象會導致細胞的損傷，因此轉位作用的調控極為重要。反轉錄病毒（retrovirus）的 Integrase （核酸銜接酵素）所扮演的角色是將反轉錄酵素所反轉錄之 cDNA 銜接嵌入至寄主細胞的標的 DNA (target DNA)基因組中，在銜接嵌入過程中也有著類似的轉位機轉。
對整個 IS3 family而言，N 端在胺基酸序列上較不保守，但均有 helix-turn-helix DNA binding domain ; C 端有一個核心催化區 (catalytic core domain)，含有活化催化位置 DDE motif的結構，負責轉位機制（transposition）過程中 DNA 的銜接/瓦解的所有催化反應。其催化反應中心具有 Asp 及 Glu 兩個殘基 (residue)，可以與二價金屬離子結合配位，維持催化中心的活性。本篇研究利用了電腦模擬的方法，利用現有的理論、資料庫及軟體（InsightII），建立 ISLC3 的 C 端核心催化區的分子模型，建立分子模型的資料，將有助於分子結構和其功能關係之研究，對 ISLC3 轉位子分子機制將能有更深入的瞭解。

ISLC3 is a member of IS3 family that is isolated from Lactobacillus casei. It has 1,351 bp and encodes for 382 amino acids. ISLC3 contains two open reading frames, named orfA and orfB, respectively. IS3 family can be found in both Gram positive and Gram negative bacteria genomes, and it replicates in a no conservative way. Transposon is a good mutagen that is related to DNA recombination or insertion of plasmid DNA into genome. Since it has an ability of reshaping the genome, it plays a critical role in evolutionary genetics. However, the recombination of genome with high frequent transposition can be a harm to the host, so the regulation of transposition is necessary. The integrase of retrovirus plays a role in insertion of reverse-transcripted cDNA into the target DNA in host genome,
and the process of insertion is similar to transposition.
The N terminal of IS3 family is relatively nonconserved, but all contain helix-turn-helix DNA binding domain. The catalytic core domain in C terminal contains the active site, DDE motif, function in linkage/terminal all catalytic reactions. The two residues, Asp and Glu, in the catalytic site are coupled to a divalent metal ion that is essential to its activity. I used a computer stimulation method with current theory, database and software InsightII, to constructed the molecular model of C terminal catalytic core domain of ISLC3. The molecular model of ISLC3 helps to study on the relationship between molecular structure and function in order to understand
the mechanism of ISLC3 transposition.

目 錄
摘要………….…………………………..………………………………1
Abstract………….…………………………..…………………………2
第一章、簡介………….…………………………..…………………..3
1-1 ISLC3 簡介………………. ….……………..…………………...3
1-2 預測蛋白質結構的方法…………………………………………..6
1-3 蛋白質分子結構的特徵………..………………………………...8
1-4 分子動力模擬(MolecularDynamics ,MD ) …………..…….….10
第二章 實驗原理和方法…..……..………………………………..12
2-1 同源模擬法預測蛋白質分子結構……………………..…….….12
2-2 InsightII 軟體……..…………………………………...…..….16
2-3 模擬與二價金屬離子結合配位……………..…………………...18
2-4 分子動力模擬原理………………………………………………..19
第三章 結果……..…….………..…….…………………………….…29
第四章 討論……..….…………………………………………………45
第五章 參考資料 ……………………………………………………46
附錄一 HFR、HMM 模版結果……... ..……………………………………53
表目
表、一 由HFR篩選出的模版資料………………………………………29
表、二 由HMM篩選出的模版資料 ………………………………………30
表、三 ISLC3的核心催化區對模版的序列百分比 ……………………31
表、四 模版所 Model 出結構的RMSD值比較 …………………………31
表、五 ISLC3 結構Free Mg2+及Mg2+結構的構形能比較………………37
表、六 D1D2E motif，D1各原子與二價金屬離子的距離（Å）…………42
表、七 D1D2E motif，D2各原子與二價金屬離子的距離（Å）…………43
表、八 D1D2E motif，E各原子與二價金屬離子的距離（Å）…………44
圖目
圖、一 1asu、1bi4C、1bisB、1c0m、1bl3C及2itg 經由 SWISS-MODEL
Protein Modelling Server 所預測出的結構………………33
圖、二（A）ISLC3 的 C 端核心催化區與模版的序列比對……………34
（B）為紅線區為所預測出結構的序列部分 ……………………34
圖、三ISLC3 核心催化區結構與ISLC3 結構的Free Mg2+和Mg2+結構立體圖……………………………………………………………36
圖、四Hiv-1 Integrase Core Domain Complexed With Mg2+（1BIU），DDE motif 各原子與Mg2+的距離（Å）………………………………38
圖、五Crystal Structure Of Tn5 Transposase Complexed With Mn2+（1F3I），DDE motif 各原子與Mn2+的距離（Å）…………………39
圖、六Catalytic core domain of ISLC3 Complexed With Mg2+，DDE motif 各原子與Mg2+的距離（Å）………………………………40
圖、七DDE motif，各胺基酸分子的原子名稱 …………………………41

（1）.Rief, H. J., Saedler, H., IS1 is involved in deletion formation in the gal region of E. coli K12, Molecular & General Genetics, 137:17-28, 1975.
（2）.Cornelis, G., Saedler, H., Deletion and an inversion induced by a resident IS1 of the lactose transposon Tn951, Molecular & General Genetics, 178:367-74, 1980
（3）.Galas, D. J., Chandler, M., Structure and stability of Tn9-mediated cointegrates. Evidence for two pathways of transposition, Journal of Molecular Biology, 154:245-72, 1982.
（4）.Bernardi, F., Bernardi, A., Transcription of the target is required for IS102 mediated deletions, Molecular & General Genetics, 212:265-70, 1988.
（5）. Mahillon, J., and Chandler, M. 1998. Insertion sequences. Microbiol. Mol. Biol. Rev. 62:725-774.
（6）. Galas, D. J., Chandler, M., Bacterial insertion sequences. Berg, D. E., Howe, M. M., (eds) Mobile DNA, American Society for Microbiology, Washington DC, pp. 109-162, 1989.
（7）. Spielmann-Ryser, J., Moser, M., Kast, P., Weber, H., Factors determining the frequency of plasmid cointegrate formation mediated by insertion sequence IS3 from Escherichia coli, Molecular & General Genetics, 226:441-8, 1991.
（8）.Sekine, Y., Eisaki, N., Ohtsubo, E., Translational control in production of transposase and in transposition of insertion sequence IS3, Journal of Molecular Biology, 235:1406-20, 1994.
（9）. Fayet, O., Ramond, P., Polard, P., Prere, M. F., Chandler, M., Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences, Molecular Microbiology, 4:1771-7, 1990.
（10）. Polard, P., Prere, M. F., Chandler, M., and Fayet, O. 1991. Programmed transcriptional frameshifting and inition at an AUU codon in gene expression of bacterial insertion sequence IS911. J.Mol. Biol. 222:465-477.
（11）. Hu, S. T., Hwang, J. H., Lee, L. C., Lee, C. H., Li, P. L., and Hsieh, Y. C. 1994.Functional analysis of the 14 kDa protein of insertion sequence 2. J. Mol. Biol.236:503-513.
（12）. Schwartz, E., Kroger, M., and Rak, B. 1988. IS150: distribution, nucleotide sequence and phylogenetic relationships of a new E. coli insertion element. Nucleic Acids Res. 16:6789-6802.
（13）.Grindley, N. D., Leschziner, A. E., DNA transposition: from a black box to a color monitor, Cell, 83:1063-6, 1995
（14）.Kulkosky, J., Jones, K. S., Katz, R. A., Macks, J. P., Skalka, A. M., Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposase, Molecular & Cellular Biology, 12:2331-8, 1992.
（15）.Polard, P., Chandler, M., Bacterial transposase and retroviral integrases, Molecular Microbiology, 15:13-23, 1995.
（16）.Fayet, O., Ramond, P., Polard, P., Prere, M. F., Chandler, M., Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences, Molecular Microbiology, 4:1771-7, 1990.
（17）. Khan, E., Mack, J. P., Katz, R. A., Kulkosky, J., Skalka, A. M., Retroviral integrase domains: DNA binding and the recognition of LTR sequences Nucleic Acids Research, 19: 851, 1991.
（18）. Engelman, A., Liu, Ying., Chen, H., Farzan, M., and Dyda, F. (1997).
Structure-based mutagenesis of the catalytic domain of human
immunodeficiency virus type 1 integrase. J. Virol. 71, 3507-14.
（19）.Polard, P., and Chandler, M. 1995. Bacterial transposase and retroviral integrases Mol. Microbiol. 15:13-23.
（20）.Jenkins, T. M., Engelman, A., Ghirlando, R., and Craigie, R. (1996). A soluble active mutant of HIV-1 integrase: involvement of both the core and carboxyl-terminal domains in multimerization. J. Biol. Chem. 271, 7712-7718.
（21）.Leavitt, A. D., Shiue, L., and Varmus, H. E. (1993). Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro. J. Biol. Chem. 268, 2113-2119.
（22）.van Gent, D. C., Oude Groeneger, A. A. M., and Plasterk, R. H. A. (1992).Mutational analysis of the integrase protein of Human immunodeficiency virus type 2. Prol. Natl. Acad. Sci. U. S. A. 89, 9598-9602.
（23）.Fayet, O., Ramond, P., Polard, P., Prere, M. F., and Chandler, M. (1990). Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences? Mol. Microbiol. 4, 1771-1777.
（24）.Johnson, M. D., McClure, M. A., Feng, D. F., Gray, J., and Doolittle, R. F. (1986). Computer analysis of retroviral pol genes: assignment of enzymatic functions to specific sequences and homologies with nonviral enzymes. Proc. Natl. Acad. Sci. U. S. A. 83, 7648-7652.
（25）.Kulkosky, J., Jones, K. S., Katz, R. A., Mack, J. P. G., and Skalka, A. M. (1992). Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral / retrotransposon integrases and bacterial insertion sequence transposases. Mol. Cell. Biol. 12, 2331-2338.
（26）.Engelman, A., and Craigie, R. (1992). Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro. J. Virol. 66, 6361-6369.
（27）.Dyda, F., Hichman, A. B., Jenkins., T. M., Engelman, A. Craigie, R., and Davies, D. R. (1994). Crystal structure of the catalytic domain of HIV-1 integrase:similarity to other polynucleotidyl transferases. Science 266, 1981-1986.
（28）.Bujacz, G., Jaskolski, M., Alexandratos, J., Wlodawer, A., Merkel, G., Katz, R. A., and Skalka, A. M. (1995). High-resolution structure of the catalytic domain of avian sarcoma virus integrase. J. Mol. Biol. 253, 336-346.
（29）.Douglas R. Davies, Igor Y. Goryshin, William S. Reznikoff, Ivan Rayment * Three-Dimensional Structure of the Tn5 Synaptic Complex Transposition IntermediateSCIENCE Volume 289, Number 5476, Issue of 7 Jul 2000, pp. 77-85.
（30）. Mizuuchi, K. (1992). Polynucleotidyl transfer reactions in transpositional DNA recombination. J. Biol. Chem. 267, 21273-21276.
（31）Allen, M. P. Tildesley, D. J. Computer Simulation of Liquids, Clarendon Press：Oxford，1987.
（32）Ciccotti, G.；Frenkel，D.；McDonald, I. R.（Eds）Simulation of Liquids and Solids, North-Holland：Amsterdam, 1987.
（33）Ciccotti, G.；Hoover, W. G. Molecular-Dynamics Simulation of Statistical-Mechanical Systems, North-Holland：Amsterdam, 1986.
（34）Goodfellow, J. M.（Ed.）Molecular Dynamics Applications in Molecular Biology, Boca Raton：CRC Press, Inc. , 1990.
（35）Van Gunsteren, W. F.；Berensen, H. J. C. Angew. Chem. Int. Ed. Engl., 1990, 29, 992.
（36）Binder, K.；Kalos, M. H.（Eds.）Monte Carlo Methods in Statistical Physics, Springer：Berlin, 1979.
（37）Landau, D. P.；Mon, K. K.；Schuttler, H.-B. Computer Simulation Studies in Condensed Matter Physics Ⅲ, Springer-Verlag：Berlin, 1991.
（38）.Alder, B. J.；Wainwright, T. E. J. Chem. Phys. , 1957, 27, 1208.
（39）.McCammon, J. A.；Gelin, B. R.；Karplus, M. Nature, 1977, 267, 585.
（40）.Rost B. TOPITS: threading one-dimensional predictions into three-dimensional structures. In: C Rawlings, D Clark, R Altman, L Hunter, T Lengauer, & S Wodak, editors. The third international conference on Intelligent Systems for Molecular Biology (ISMB). Cambridge, U.K.: AAAI Press; 1995. p 314-321.
（41）.Fischer D, Eisenberg D. Fold recognition using sequence-derived predictions. Protein Science 1996; 5: 947-955.
（42）.Rice D., Eisenberg D. A 3D-1D substitution matrix for protein fold recognition that includes predicted secondary structure of the sequence. J Mol Biol 1997; 267: 1026-1038.
（43）.Kelley LA, MacCallum RM, Sternberg MJE. Enhanced genome annotation using structural profiles in the program 3D-PSSM. J Mol Biol 2000; 299(2): 501-522.
（44）.Fischer D. Hybrid fold recognition: combining sequence derived properties with evolutionary information. Maun Lani, HI: Pacific Symp Biocomputing, 2000. p 119-130.
（45）.Jones DT, Taylor WR, Thornton JM. A new approach to protein fold recognition. Nature 1992; 358: 86-89.
（46）.Jones DT, Miller RT, Thornton JM. Successful protein fold recognition by optimal sequence threading validated by rigorous blind testing. Proteins 1995; 23: 387-397.
（47）.Karplus K, Barrett C, Hughey R. Hidden Markov Models for detecting remote protein homologies. Bioinformatics 1998; 14(10): 846-856.
（48）.Guex N, Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 1997; 18: 2714-2723
（49）.Bushman, F. D., and Craigie, R. (1990) Sequence requirements for integration of Moloney murine leukemia virus DNA in vitro. J. Virol. 64, 5645-5648.
（50）.Ellison, V., Abrams, H., Roe, T., Lifson, J., and Brown, P. O. (1990). Human immunodeficiency virus integration in a cell-free system. J. Virol. 64, 2711-2715.
（51）.Fitzgerald, M. L., Vora, A. C., Zeh, W. C., and Grandgenett, W. P. (1992). Concerted integration of viral DNA termini by purified Avian myeloblastosis virus integrase. J. Virol. 66, 6257-6263.
（52）.Grandgenett, D. P., and Mumm, S. R. (1990). Unraveling retrovirus integration. Cell 60, 3-4.
（53）.Bukrinsky, M. I., Sharova, N., McDonald, T. L., Pushkarskaya, T., Tarpley, W. G., and Stevenson, M. (1993). Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proc. Natl. Acad. Sci. U. S. A. 90, 6125-6129.
（54）.Farnet, C., and Haseltine, W. A. (1991). Determination of viral proteins present in the Human immunodeficiency virus type 1 preintegration complex. J. Virol. 65, 1910-1915.
（55）.Gulizia, J., Dempsey, M. P., Sharova, M., Bukrinsky, M. I., Spitz, L., Goldfarb, D., and Stevenson. M. (1994). Reduced nuclear import of HIV-1 preintegration complexes in the presence of a prototypic nuclear
targeting signal. J. Virol. 68, 2021-2025.
（56）.Lapadat-Tapolski, M., De Rocquigny, H., Van Gent, D. C., Rocques, B., Plasterk, R. H. A., and Darlix, J. L. (1993). Interactions between HIV-1 nucleocapsid protein and viral DNA may have important functions in the viral life cycle. Nucleic Acids Res. 21, 831-839.
（57）.Vink, C., Oude Groeneger, A. A. M., and Plasterk, R. H. A. (1993). Identification of the catalytic and DNA-binding region of human immunodeficiency virus type 1 integrase protein. Nucleic Acids Res. 21, 1419-1425.
（58）.Bernstein FC, Koetzle TF, Williams GJ, Meyer, Jr., EE, Brice MD, Rodgers JR, Kennard O, Shimanouchi T, Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol 1977; 112: 535-542.
（59）.Vink, C., Oude Groeneger, A. A. M., and Plasterk, R. H. A. (1993). Identification of the catalytic and DNA-binding region of human immunodeficiency virus type 1 integrase protein. Nucleic Acids Res. 21, 1419-1425.
（60）.Engelman, A., Liu, Ying., Chen, H., Farzan, M., and Dyda, F. (1997). Structure-based mutagenesis of the catalytic domain of human immunodeficiency virus type 1 integrase. J. Virol. 71, 3507-14.
（61）.Lovell, S., Goryshin, I. Y., Reznikoff, W. R., Rayment, I.: Two-Metal Active Site Binding of a Tn5 Transposase Synaptic Complex Nat.Struct.Biol. 9 pp. 278 (2002)
（62）.Lubkowski, J., Dauter, Z., Yang, F., Alexandrator, J., Merkel, G. Skalka, A. M., and Wlodawer, A. (1999). Atomic resolution structures of the core domain of avian sarcoma virus integrase and its D64N mutant. Biochemistry 38, 13512-13522.
（63）.Freemont, P. S., Friedman, J. M., Beese, L. S., Sanderson, M. R., and Steitz, T. A. (1988). Cocrystal structure of an editing complex of Klenow fragment with DNA. Proc. Natl. Acad. Sci. U. S. A. 85, 8924-8928.
（64）.Beese, L. S., and Steitz, T. A. (1991). Structural basis for the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J. 10, 25-33. 65.van Gent, D.C., Elgersma, Y., Bolk, M. W. J., Vink, C., and Plasterk, R. H. A. (1991). DNA binding properties of the integrase proteins of Human immunodeficiency virus type 1 and 2. Nucleic Acids Res. 19, 3821-3827.
（65）L. M. Braam, I. Y. Goryshin, W. S. Reznikoff, J. Biol. Chem. 274, 86 (1999)
（66）.Vink, C., Oude Groeneger, A. A. M., and Plasterk, R. H. A. (1993). Identification of the catalytic and DNA-binding region of human immunodeficiency virus type 1 integrase protein. Nucleic Acids Res. 21, 1419-1425.
（67）. Kahn, E., Mack, J. P. G., Katz, R. A., Kulkosky, J., and Skalka, A. M. (1991). Retroviral integrase domains: DNA binding and the recognition of LTR sequences. Nucleic Acids Res. 19, 851-860. 68. Bushman, F. D., Engelman, A., Palmer, L., Wingfieid, P., and Craigie, R. (1993). Domains of the integrase protein of human immunodeficiency virus type 1 responsible for polynucleotidyl transfer and Zinc binding. Proc. Natl. Acad. Sci. U. S. A. 90, 3428-3432.
（69）.Dyda, F., Hichman, A. B., Jenkins., T. M., Engelman, A. Craigie, R., and Davies, D. R. (1994). Crystal structure of the catalytic domain of HIV-1 integrase:similarity to other polynucleotidyl transferases. Science 266, 1981-1986.
（70）.Engelman, A., and Craigie, R. (1992). Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro. J. Virol. 66, 6361-6369.
（71）.Kulkosky, J., Jones, K. S., Katz, R. A., Mack, J. P. G., and Skalka, A. M. (1992). Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral / retrotransposon integrases and bacterial insertion sequence transposases. Mol. Cell. Biol. 12, 2331-2338.
（72）.Leavitt, A. D., Shiue, L., and Varmus, H. E. (1993). Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro. J. Biol. Chem. 268, 2113-2119.
（73）.van Gent, D. C., Oude Groeneger, A. A. M., and Plasterk, R. H. A. (1992). Mutational analysis of the integrase protein of Human immunodeficiency virus type 2. Prol. Natl. Acad. Sci. U. S. A. 89, 9598-9602.
（74）. Vink, C., Oude Groeneger, A. A. M., and Plasterk, R. H. A. (1993). Identification of the catalytic and DNA-binding region of human immunodeficiency virus type 1 integrase protein. Nucleic Acids Res. 21, 1419-1425.
（75）Kreyszig, E. Advance Engineering Mathematics, John Wiley & Sons：New York, 1988.
（76）Verlet, L. Phys. Rew. , 1967, 159, 98.
（77）Allen, M. P.；Tildesley，D. J. Computer Simulation of Liquids, Oxford Sci. Publ.：New York, 1987.
（78）Beenman, D. J. J. Comput. Phys. , 1976, 20, 130.
（79）Allen, M. P. Tildesley, D. J. Computer Simulation of Liquids, Clarendon Press：Oxford, 1987.