臺灣博碩士論文加值系統

兩種因紫外光照射而造成DNA損傷的產物主要為CPDs與6-4PPs，其造成DNA結構的變形，而導致DNA轉錄（transcription）與複製（replication）受阻。而紫外光損傷DNA也是探討細胞內可辨識DNA結構扭曲蛋白之良好探針。本實驗為探討脊椎動物胚胎發育過程中，損傷DNA辨識因子之表現，利用斑馬魚發育中之胚胎為系統模式，並以紫外光損傷DNA為辨識基質進行實驗。以受精後12至84小時的斑馬魚胚胎萃取液進行凝膠阻抗試驗，發現隨著胚胎發育時間的演進，12小時胚胎蛋白產生的高分子量辨識複合體，而84小時胚胎蛋白則產生低分子量辨識複合體。以抗人類修補辨識蛋白XPA、RPA-70、RPA-32抗體進行西方墨點法，發現胚胎發育12小時至60小時中，這些辨識蛋白都不表現，而發育至84小時後XPA、RPA-32始可測得，故胚胎早期所含之辨識蛋白應非與人類DNA損傷辨識蛋白相似者。再將84小時胚胎萃取液通過親和性吸附分析發現，84小時所表現的類XPA具有辨識紫外光損傷DNA之活性，因此XPA應參與此時期的損傷辨識作用。分析12小時胚胎萃取液特性之實驗發現，其對NaCl耐受性較84小時萃取液為低，約0.1 M，而競爭性實驗發現12小時萃取液以相同的辨識蛋白群對CPD或6-4PPs進行辨識。透過雙向電泳的分析得知，12小時萃取液的辨識蛋白群其pI值大約集中於7.5至10之間，屬於略呈鹼性的蛋白群。再將12小時萃取液辨識複合體其中之二個polypeptide進行蛋白質質譜儀析與mascot資料庫比對得知，polypeptides 50.2-kDa與29-kDa分別與斑馬魚fast skeletal muscle troponin T與vitellogenin有高度相似。

The expression of distorted DNA-binding factors was studied in developing zebrafish using UV-damaged DNA as the binding target. A strong and high-shifting binding activity was detected in the extracts of zebrafish embryo (12 h after fertilization), and the expression of the activity dramatically decreased in 60 to 84-h-old zebrafish. Unlike the embryonic extract, the extracts of 84-h-old larvae produced only low-shifting binding complexes when incubated with a CPD-specific probe. The binding of 12-hr-old and 84-hr-old extracts to CPD- and 6-4PPs under various concentration of NaCl supported the possibility that different damage-recognition protein were expressed in zebrafish according to their developmental condition. A competitive binding assay indicated that both CPDs and 6-4PPs were recognized by the same binding activity expressed in 12-h-old zebrafish. The binding activity expressed in embryos was apparently unrelated to human DNA repair damage-recognition proteins XPA、RPA-70 and RPA-32, since those polypeptides recognized by an anti-human XPA、RPA-70 and RPA-32 antibody were detected only in 84-h-old zebrafish extracts. Identification of one 50.2 and one 29-kDa UV-damage-DNA binding factor in 12-hr-old zebrafish extracts by EMSA linked to SDS-PAGE and determined by MALDI TOF analysis, indicated that the 29-kDa and 50.2-kDa polypeptides are high homologous to zebrafish fast skeletal muscle troponin T and vitellogenin. UV-binding factors found in 12-h-old zebrafish embryos may be involved in processing developmental stage-specific DNA structures similar to UV-damage DNA.

目次
中文摘要………………………………………………………………..01
英文摘要………………………………………………………………..02
續論……………………………………………………………………..03
壹、DNA結構變異的形成因子及其影響………………………….03
貳、細胞中各類DNA損傷辨識蛋白特性分析…………………….05
參、研究目的…………………………………………………….…09
材料與方法………………………………………………………….….11
壹、材料……………………………………………………………11
貳、實驗方法………………………………………………………20
結果…………………………………...………………………………..32
討論……………………………………………...……………………..36
參考文獻……………………………………………………...………..42
附圖…………………………………………………...………………..47

Boulikas, T. 1996. DNA lesion-recognizing proteins and the p53 connection. Anticancer Res. 16:225-242.
Bucher, E. A., Dhoot, G. K., Emerson, M.M., Ober, M. 1999. Structure and evolution of the alternatively spliced fast troponin T isoform gene. J. of Biol. Chem. 274(25):17661-70.
Carell, T., Burgdorf, L. T., Kundu, L. M., Cichon, M. 2001. The mechanism of action of DNA photolyases. Current Opin. Chem. Biol. 5(5):491-8.
Cleacer, J.E., States, J.C. 1997. The DNA damage-recognition problem in human and other eukaryotic cell: the XPA damage binding protein. Biochem. J. 321 : 1-12.
Falchuk, K.H. 1998. The molecular basis for the role of zinc in developmental biology. Mol. Cellu. Biochem. 188(1-2) : 41-8.
Fujiwara, Y.C., Masutani, T., Mizukoshi, J., Kondo, F., Hanaoka, S. 1999. Characterization of DNA recognition by the human UV-damaged DNA-binding protein. J. Biol. Chem. 274:20027-33.
Gadducci, A., Cosio, S., Cionini, L., Genazzani, A. R. 2001. Neoadjuvant chemotherapy and concurrent chemoradiation in the treatment of advanced cervical cancer. Anticancer Res. 21(5):3525-33.
Guzder, S. N., Sung, L., Prakash, S. 1996. Nucleotide excision repair in yeast is mediated by sequential assembly of repair factors and not by a pre-assembled repairosome. J. Biol. Chem. 271:8903-8910.
Guzder, S. N., Habraken, Y., Sung, P., Prakash, L., Prakash, S. 1995. J. Biol. Chem. 270:12973-12976.
Hayes, S., Shiyanov, P., Chen, X., Raychaudhuri, P. 1998. DDB, a putative DNA repair protein, can function as a transcriptional partner of E2F1. Mol. Cell Biol. 18:240-249.
Huang, J. C., Zamble, D.B., Reardon, J.T., Lippard, S.J., Sancar, A. 1994. HMG-domain proteins specifically inhibit the repair of the major DNA adduct of the anticancer drug cisplatin by human excision nuclease. Proc. Natl. Acad. Sci. U.S.A. 91:10394-10398.
Ikegami, T., Kuraoka, I., Saijo, M., Kodo, N., Kyogoku, Y., Morikawa, K.,Tanaka, K., Shirakawa, M. 1999. Resonance assignments, solution structure, and backbone dynamics of the DNA-and RPA-binding domain of human repair factor XPA. J. Biochem. 125 : 495-506.
Islas, A.L., P.C., Hanawalt. 1995. DNA repair in the MYC and FMS proto-oncogenes in ultraviolet light-irradiated human HL60 promyelocytic cells during differentiation. Cancer Res. 55:336-341.
Iyer, R., Lehnert, B.E. 2000. Effects of ionizing radiation in targeted and nontargeted cells. Archives. Biochem. Biophys. 376:14-25.
Jackson, S.P. 1996. The recognition of DNA damage. Cur. Opin. Gen. Dev. 6:19-25.
Kai, M., Takahashi, T.T., Todo, K., Sakaguchi. 1995. Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster. Nucleic Acids Res. 23:2600-2607.
Kai, M., Todo, T.M., Wada, H., Ryo, C., Masutani, H., Kobayashi, H., Morioka, E., Ohtsuka, F., Hanaoka, K., Sakaguchi. 1998. A new Drosophila ultraviolet light-damaged DNA recognition endonuclease that selectively nicks a (6-4) photoproduct site. Biochim. Biophys. Acta. 1397(2):180-188.
Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F. 1995. Stages of embryonic development of the zebrafish. Dev. Dyn. 203:253-310.
Latimer, J.J., Hultner, M.L., Cleaver, J.E., Pedersen, R.A.1996. Elevated DNA excision repair capacity in the extraembryonic mesoderm of the midgestation mouse embryo. Experimental Cell Res. 228:19-28.
Lydall, D., Weinert, T. 1996. From DNA damage to cell cycle arrest and suicide: a budding yeast perspective. Cur. Opin. Gen. Dev. 6:4-11.
Mu, D., Hsu, D.S., Sancar, A. 1996. Reaction mechanism of human DNA repair excision nuclease. J. Biol. Chem. 271:8285-8294.
Nagaki, S., Yamamoto, M., Yumoto, Y., Shirakawa, H., Yoshida, M., Teraoka, H. 1998. Non-histone chromosomal proteins HMG1 and 2 enhance ligation reaction of DNA double-strand breaks. Biochem. Biophys. Res. Commun. 246:137-141.
Nichols, A.F., Itoh, T., Graham, J.A., Liu, W., Yamaizumi, M., Linn, S. 2001. Human damage-specific DNA-binding protein p48. Characterization of XPE mutations and regulation following UV irradiation. Journal of Biological Chemistry. 275(28):21422-8.
Nichols, A.F., Ong, P., Linn, S. 1996. Mutations specific to the xeroderma pigmentosum group E Ddb-phenotype. J. Biol. Chem. 271:24317-24320.
Nordberg, J., Arner, E.S. 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radical Biology & Medicine. 31(11):1287-312.
Otrin, V.R., McLenigan, M., Takao, M., Levine, A.S., Protic, M. 1997. Translocation of a UV-damaged DNA binding protein into a tight association with chromatin after treatment of mammalian cells with UV light. J. Cell Sci. 110:1159-1168.
Pasheva, E.A., Pashev, I.G., Favre, A. 1998. Preferential binding of high mobility group 1 protein to UV-damaged DNA. J. Biol. Chem. 273:24730-24736.
Patrick, S.M., Turchi, J.J. 1998. Human replication protein A preferentially binds cisplatin-damaged duplex DNA in vitro. Biochemistry. 37:8808-8815.
Patrick, S.M., Henkels, K.M., Turchi, J.J. 1997. High-mobility group 1 protein inhibits helicase catalyzed displacement of cisplatin-damaged DNA. Biochimica et Biophysica Acta. 1354:279-290.
Richards, M.P. 1997. Trace mineral metabolism in the avian embryo. Poultry Sci. 76(1) : 152-64.
Seidl, C., Moritz, K.B. 1998. A novel UV-damaged DNA binding protein emerges during the chromatin-eliminating cleavage period in Ascaris suum. Nucl. Acids Res. 26:768-777.
Shiyanov, P., Hayes, S.A., Donepudi, M., Nichols, A.F., Linn, S., Slagle, B.L., Raychaudhuri, P. 1999. The naturally occurring mutants of DDB are impaired in stimulating nuclear import of the p125 subunit and E2F1-activated transcription. Mol. Cell. Biol. 19:4935-4943.
Sugasawa, K., Masutani, C., Iwai, S., van der Spek, P.J., Eker, A.P., Hanaoka, F. Bootsma D. Hoeijmakers J.H. 1998. Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair. Mol. Cell. 2:223-232.
Thoma, F. 1999. Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair. EMBO J. 18:6585-6598.
Tornaletti, S., Pfeifer, GP. 1996. UV damage and repair mechanisms in mammalian cells. Bioessays. 18(3):221-8.
Tuteja, N., Singh, M.B., Misra, M.K., Bhalla, P.L., Tuteja, R. 2001. Molecular mechanisms of DNA damage and repair: progress in plants. Crit. Rev. Biochem. Mol. Biol. 36(4):337-97.
Vichi, P., Coin, F., Renaud, J-P.,Vermeulen, W., Hoeijmarkers, J.H.J., Moras, D., Egly, J.M. 1997. Cisplatin-and UV-damage DNA lure the basal transcription factor TFIID/TBP. EMBO J. 16 : 7444-7456.
Wakasugi, M., Sancar, A. 1998. Assembly, subunit composition, and footprint of human DNA repair excision nuclease. Proc. Natl. Acad. Sci. USA. 95:6669-6674.
Wakasugi, M., Sancar, A. 1999. Order of assembly of human DNA repair excision nuclease. J. Biol. Chem. 274:18759-18768.
Wakasugi, M., Abe, Y., Yoshida, Y., Matsunaga, T., Nikaido, O. 1996. Purification of a novel UV-damage-DNA binding protein highly specific for (6-4) photoproduct. Nucl. Acids Res. 24:1099-1104.
Wang, J.F., Bashir, M., Engelsberg, B.N., Witmer, C., Rozmiarek, H., Billings, P.C. 1997. High mobility group proteins 1 and 2 recognize chromium-damaged DNA. Carcinogenesis. 18:371-375.
Wood, R.D. 1996. DNA repair in eukaryotes. Annu. Rev. Biochem. 65:135-167.
Xu, Y., He, J., Wang, X., Lim, TM., Gong, Z. 2000. Asynchronous activation of 10 muscle-specific protein (MSP) genes during zebrafish somitogenesis. Developmental Dynamics. 219(2):201-15.