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研究生:陳盈坤
研究生(外文):Ying-Kun Chen
論文名稱:核酸錯誤配對及核酸環修復之反應區段分析
論文名稱(外文):Repair Patch Analysis of DNA Mismatch and Loop Repair
指導教授:方偉宏方偉宏引用關係
指導教授(外文):Woei-Horng Fang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫事技術學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:57
中文關鍵詞:修復路徑移除反應配對錯誤核酸環修復DNA修復核酸修復
外文關鍵詞:mismatch repairrepair patchDNA repairthiophosphate chemistryLoop repairexcision mapping
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演化過程中所保留下來的MMR可以藉由修復DNA複製錯誤,抑制發生在部分解離DNA序列之間的非同質性重組,與不同的DNA損害作用以及一些其他的功能來維持真核與原核生物基因體的穩定性。在核酸配對錯誤復上已有相當透澈的瞭解。但是大型核酸環的修復,一個迥異於配對錯誤的修復機制,卻是個未知的系統。因此研究這兩種修復系統間的差異,將是個令人感興趣的議題。
人類特異股配對修復及大型核酸環修復均需外加dNTP,同時修復反應也受到aphidicolin的抑制。之前關於配對錯誤修復片段的研究多半是在HeLa細胞核萃取物修復異雙股核酸反應中,不外加dNTP或加入aphidicolin的條件下,截取修復反應的中間產物。於試管中停止再合成反應的情況下,觀察移除反應後斷裂的單股核酸產物,以進行移除反應範圍的分析。因此觀察到移除反應區域橫跨在錯誤至斷端這範圍。然而這類型的研究方法沒有使修復反應作用完全,因此不能排除所得結果是來自中止反應所衍生副反應的訊號。
為了避免此項疑慮,因此改用含硫磷酸的化學方法來探討可能修復路徑。利用dNTPαS和iodoethanol處理下,核酸配對錯誤、核酸環、或兩者同時存在下的3’含斷股之異雙股核酸呈現出個別的修復路徑。在核酸配對錯誤修復方面,最遠的取代訊號出現在距離錯誤配對5’端100~150 鹼基長度的位置。這與一般移除反應路徑分析終止端位置結果一致。在核酸環修復方面,修復的訊號則僅位於斷端與核酸環兩點。因此推論:核酸配對錯誤與核酸環修復是經由不同的機制修復,然而當核酸配對錯誤與核酸環共同存在時,則會影響彼此的修復反應。
Evolutionarily conserved mismatch repair (MMR) systems promote genetic stability in most eukaryotic and prokaryotic organisms by correcting DNA replication errors, antagonizing homeologous recombination between partially diverged DNA sequences, interacting with variety of DNA lesions, and performing other functions. The study about mismatch repair has been analyzed thoroughly. But the large loop repair, a distinct repair from mismatch repair, is an unknown repair system. Studying the differences between these two repair systems seems to be an interesting project.
Both human strand-specific mismatch repair and large loop repair depend on presence of exogenous dNTPs and are inhibited by aphidicolin. Most of previous studies about mismatch repair patches were to trap the intermediates produced from the course of heteroduplex correction in HeLa nuclear extracts either in the absent of exogenous dNTP or in the supplement of aphidicolin. In vitro excision tracts have been mapped as single-strand gaps produced when repair is provoked under condition of restricted DNA synthesis. Analysis of gaps produced on these heteroduplex resulted in excision tracts spanning the path between lesion and the nick. However, according to this methodology, this repair reaction is incomplete. It is not able to eliminate the possibility of the result generated from side reaction caused by the restricted to show that condition.
To avoid the argument, we employed thiophosphate chemistry in the repair patch mapping. Under the condition of dNTPΠ substitution and iodoethanol treatment, mismatch, large loop, or mismatch and large loop co-exist heteroduplexses with 3’ nick showed distinct repair patches. In mismatch repair, the most extreme ladders shift to 5’ direction approximately 100 to 150 bases beyond the mismatch, which is consistent with the excision tract endpoint mapped and agrees with the results of others. In loop repair, the repair signal locates on nick and loop site. These results further ascertain that the repair of mismatch and large loop are processed through different mechanisms. When mismatch and loop co-exist, there is interference between these two repair patches.
總目次 I
表目次 III
圖目次 IV
附錄目次 V
中文摘要 VI
英文摘要 VII
縮寫表 IX

前言 1
材料與方法 8
一、菌株 8
二、人類細胞株之繼代培養 8
三、桿菌細胞萃取物之製備 9
四、人類細胞核萃取液之製備 10
五、突變噬菌體M13mp18 mutant、f1PM mutant之建構 11
六、M13mp18與f1PM系列複製型雙股核酸及單股核酸之製備 12
七、異雙股核酸之建構 14
八、異雙股核酸對測定用限制酵素之敏感度分析 15
九、試管中反應條件之選定與修復反應 16
十、試管中修復情形之分析 17
十一、γ-P32[ATP]標記探針 17
十二、電泳分析核酸產物 18
十三、南方墨點分析 19
十四、核酸產物分析 19
結果 21
一、具斷股異雙股核酸之建構 21
二、異雙股核酸對分析用限制酵素之敏感度分析 21
三、試管中修復反應條件之選定 22
四、試管中異雙股核酸之修復反應定量用限制酵素之
敏感度分析 23
五、在大腸桿菌萃取液以及人類細胞核萃取液中添加
dCTPαS對修復反應之影響 24
六、鹼性瓊脂凝膠電泳分析 25
七、8.3 M尿素-5%聚丙醯胺凝膠電泳分析 26
甲、 核酸環修復與核酸配對錯誤修復 26
乙、 核酸配對錯誤與核酸環共同修復 27
討論 30
附表 33
附圖 36
附錄 47
參考文獻 52
Abuin, A., Zhang, H., and Bradley, A. (2000). Genetic analysis of mouse embryonic stem cells bearing Msh3 and Msh2 single and compound mutations. Mol Cell Biol 20, 149-157.
Ayares, D., Ganea, D., Chekuri, L., Campbell, C. R., and Kucherlapati, R. (1987). Repair of single-stranded DNA nicks, gaps, and loops in mammalian cells. Mol Cell Biol 7, 1656-1662.
Balmain, A., and Birnie, G. D. (1979). Nick translation of mammalian DNA. Biochim Biophys Acta 561, 155-166.
Bellacosa, A. (2001). Functional interactions and signaling properties of mammalian DNA mismatch repair proteins. Cell Death Differ 8, 1076-1092.
Buermeyer, A. B., Deschenes, S. M., Baker, S. M., and Liskay, R. M. (1999). Mammalian DNA mismatch repair. Annu Rev Genet 33, 533-564.
Carraway, M., and Marinus, M. G. (1993). Repair of heteroduplex DNA molecules with multibase loops in Escherichia coli. J Bacteriol 175, 3972-3980.
Chambers, S. R., Hunter, N., Louis, E. J., and Borts, R. H. (1996). The mismatch repair system reduces meiotic homeologous recombination and stimulates recombination-dependent chromosome loss. Mol Cell Biol 16, 6110-6120.
Connelly, J. C., de Leau, E. S., Okely, E. A., and Leach, D. R. (1997). Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli. J Biol Chem 272, 19819-19826.
Connelly, J. C., Kirkham, L. A., and Leach, D. R. (1998). The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA. Proc Natl Acad Sci U S A 95, 7969-7974.
Connelly, J. C., and Leach, D. R. (1996). The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination. Genes Cells 1, 285-291.
Cooper, D. L., Lahue, R. S., and Modrich, P. (1993). Methyl-directed mismatch repair is bidirectional. J Biol Chem 268, 11823-11829.
Corrette-Bennett, S. E., Parker, B. O., Mohlman, N. L., and Lahue, R. S. (1999). Correction of large mispaired DNA loops by extracts of Saccharomyces cerevisiae. J Biol Chem 274, 17605-17611.
Deng, W. P., and Nickoloff, J. A. (1994). Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells. Mol Cell Biol 14, 400-406.
Fang, W., Wu, J. Y., and Su, M. J. (1997). Methyl-directed repair of mismatched small heterologous sequences in cell extracts from Escherichia coli. J Biol Chem 272, 22714-22720.
Fang, W. H., and Modrich, P. (1993). Human strand-specific mismatch repair occurs by a bidirectional mechanism similar to that of the bacterial reaction. J Biol Chem 268, 11838-11844.
Fang, W. H., Wang, B. J., Wang, C. H., Lee, S. J., Chang, Y. T., Chuang, Y. K., and Lee, C. N. (2003). DNA loop repair by Escherichia coli cell extracts. J Biol Chem 278, 22446-22452.
Genschel, J., Littman, S. J., Drummond, J. T., and Modrich, P. (1998). Isolation of MutSbeta from human cells and comparison of the mismatch repair specificities of MutSbeta and MutSalpha. J Biol Chem 273, 19895-19901.
Grilley, M., Griffith, J., and Modrich, P. (1993). Bidirectional excision in methyl-directed mismatch repair. J Biol Chem 268, 11830-11837.
Gu, L., Hong, Y., McCulloch, S., Watanabe, H., and Li, G. M. (1998). ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair. Nucleic Acids Res 26, 1173-1178.
Harfe, B. D., and Jinks-Robertson, S. (2000). DNA mismatch repair and genetic instability. Annu Rev Genet 34, 359-399.
Holmes, J., Jr., Clark, S., and Modrich, P. (1990). Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines. Proc Natl Acad Sci U S A 87, 5837-5841.
Horst, J. P., Wu, T. H., and Marinus, M. G. (1999). Escherichia coli mutator genes. Trends Microbiol 7, 29-36.
Huang, Y. M., Chen, S. U., Goodman, S. D., Wu, S. H., Kao, J. T., Lee, C. N., Cheng, W. C., Tsai, K. S., and Fang, W. H. (2004). Interaction of nick-directed DNA mismatch repair and loop repair in human cells. J Biol Chem.(in press)
Jiricny, J. (1998). Replication errors: cha(lle)nging the genome. Embo J 17, 6427-6436.
Jiricny, J. (2000). Mediating mismatch repair. Nat Genet 24, 6-8.
Kolodner, R. (1996). Biochemistry and genetics of eukaryotic mismatch repair. Genes Dev 10, 1433-1442.
Kolodner, R. D., and Marsischky, G. T. (1999). Eukaryotic DNA mismatch repair. Curr Opin Genet Dev 9, 89-96.
Kunkel, T. A. (1993). Nucleotide repeats. Slippery DNA and diseases. Nature 365, 207-208.
Lahue, R. S., Au, K. G., and Modrich, P. (1989). DNA mismatch correction in a defined system. Science 245, 160-164.
Lahue, R. S., Su, S. S., and Modrich, P. (1987). Requirement for d(GATC) sequences in Escherichia coli mutHLS mismatch correction. Proc Natl Acad Sci U S A 84, 1482-1486.
Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000). The crystal structure of DNA mismatch repair protein MutS binding to a G x T mismatch. Nature 407, 711-717.
Littman, S. J., Fang, W. H., and Modrich, P. (1999). Repair of large insertion/deletion heterologies in human nuclear extracts is directed by a 5'' single-strand break and is independent of the mismatch repair system. J Biol Chem 274, 7474-7481.
McCulloch, S. D., Gu, L., and Li, G. M. (2003). Bi-directional processing of DNA loops by mismatch repair-dependent and -independent pathways in human cells. J Biol Chem 278, 3891-3896.
Modrich, P. (1997). Strand-specific mismatch repair in mammalian cells. J Biol Chem 272, 24727-24730.
Modrich, P., and Lahue, R. (1996). Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu Rev Biochem 65, 101-133.
Moses, R. E. (2001). DNA damage processing defects and disease. Annu Rev Genomics Hum Genet 2, 41-68.
Nakamaye, K. L., Gish, G., Eckstein, F., and Vosberg, H. P. (1988). Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-thiotriphosphates. Nucleic Acids Res 16, 9947-9959.
Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000). Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA. Nature 407, 703-710.
Palombo, F., Gallinari, P., Iaccarino, I., Lettieri, T., Hughes, M., D''Arrigo, A., Truong, O., Hsuan, J. J., and Jiricny, J. (1995). GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells. Science 268, 1912-1914.
Parker, B. O., and Marinus, M. G. (1992). Repair of DNA heteroduplexes containing small heterologous sequences in Escherichia coli. Proc Natl Acad Sci U S A 89, 1730-1734.
Radman, M. (1989). Mismatch repair and the fidelity of genetic recombination. Genome 31, 68-73.
Raschle, M., Marra, G., Nystrom-Lahti, M., Schar, P., and Jiricny, J. (1999). Identification of hMutLbeta, a heterodimer of hMLH1 and hPMS1. J Biol Chem 274, 32368-32375.
Sia, E. A., Kokoska, R. J., Dominska, M., Greenwell, P., and Petes, T. D. (1997). Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes. Mol Cell Biol 17, 2851-2858.
Su, S. S., Lahue, R. S., Au, K. G., and Modrich, P. (1988). Mispair specificity of methyl-directed DNA mismatch correction in vitro. J Biol Chem 263, 6829-6835.
Thomas, D. C., Roberts, J. D., and Kunkel, T. A. (1991). Heteroduplex repair in extracts of human HeLa cells. J Biol Chem 266, 3744-3751.
Umar, A., Boyer, J. C., and Kunkel, T. A. (1994). DNA loop repair by human cell extracts. Science 266, 814-816.
Umar, A., Buermeyer, A. B., Simon, J. A., Thomas, D. C., Clark, A. B., Liskay, R. M., and Kunkel, T. A. (1996). Requirement for PCNA in DNA mismatch repair at a step preceding DNA resynthesis. Cell 87, 65-73.
Umar, A., and Kunkel, T. A. (1996). DNA-replication fidelity, mismatch repair and genome instability in cancer cells. Eur J Biochem 238, 297-307.
Wang, H., and Hays, J. B. (2003). Mismatch repair in human nuclear extracts: effects of internal DNA-hairpin structures between mismatches and excision-initiation nicks on mismatch correction and mismatch-provoked excision. J Biol Chem 278, 28686-28693.
Wu, P. L., Lin, S. B., Huang, C. P., and Chiou, R. Y. (2002). Antioxidative and cytotoxic compounds extracted from the sap of Rhus succedanea. J Nat Prod 65, 1719-1721.
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