臺灣博碩士論文加值系統

核酸配對錯誤修復系統(DNA mismatch repair system)在生物體中扮演著維持核酸序列穩定的角色。此系統可提高複製時的忠誠度達一百至一千倍以上。目前以大腸桿菌中核酸配對錯誤修復系統－MutHLS pathway－的研究最為透徹，而參與此系統的基因產物在真核生物(酵母菌、老鼠、人類等等)中亦可以找到相對應的相似物(MutHLS homologue)。最近，在老鼠的基因剔除實驗亦開始探討這些基因相似物的生物角色，也證實了這些基因相似物和某些癌症的好發的確有關連。然而在老鼠細胞中，此系統對於不同種類的鹼基配對錯誤修復情形在試管中的生化分析上一直缺乏整體的研究。
為了評估老鼠細胞核萃取液中的核酸配對錯誤修復活性，我們以f1PM噬菌體設計了一系列含有八種單一鹼基配對錯誤的異雙股核酸，並藉由試管中修復實驗來重現反應的發生。根據實驗結果顯示，老鼠M12細胞株跟人類細胞株相當類似，都具有修復單一鹼基配對錯誤的能力，而且是以斷股(nick)來指引修復反應的發生，具有修復反應的股專一性(strand specificity)。另外，修復反應中必須要有ATP、dNTP、Mg2+的參與，再者修復反應可以被aphidicolin所抑制，但不被ddTTP所抑制，這暗示了DNA polymerase d可能參與了此套修復系統。另一個有趣的現象是，修復的效率會隨著鹼基配對錯誤處與斷股間距離的增加而降低，而無論是3’端或是5’端的斷股均會指引修復的發生。另外，八種不同的鹼基配對錯誤也各有不同的修復效率(以C-C配對錯誤的修復效率最低)，由於八種異雙股核酸的設計上除了配對錯誤以外其餘序列均相同，所以修復效率上所產生的差異應該與配對錯誤本身的特性相關，亦即代表修復系統對於不同的配對錯誤具有不同的辨識能力，因而導致了修復效率的不同。
在研究中，我們強化了單一鹼基配對錯誤異雙股核酸的設計，使八種配對錯誤均能座落在相同的序列中，避免其他序列上的差異影響了配對錯誤的修復效率，同時採用了有效的方式來評估老鼠細胞核萃取液於試管中的配對錯誤修復活性，而成功的建立了一套可信的試管中分析方法。盼望今後能在此試管中修復反應分析方法的協助下，對於老鼠配對錯誤修復活性的生化研究上，提供更多有力的資訊，進而對於徹底瞭解真核細胞的修復機制、找尋其他未知的修復蛋白，乃至於針對配對錯誤修復活性缺失與癌症好發之間的關係，能有更多的探討與認識。

We constructed a set of DNA heteroduplexes contain eight possible base pair mismatches to characterize the repair activity in mouse cell extracts. We found that cell free nuclear extracts derived form mouse M12 cell line were able to correct single base-base mispairs within open circular DNA heteroduplexes containing a strand-specific, site-specific incision. Correction in this extract system, like human mismatch repair pathway, was strand specific, and highly biased to the incised DNA strand. The in vitro activity was dependent on ATP, dNTP, Mg2+, and the distance between the incision and mismatch site. Correction of each of these heteroduplexes was abolished by aphidicolin but was relatively insensitive to the presence of ddTTP, indicating probable involvement of DNA polymerase d in this system. Different base-base mismatches within a homologous set of heteroduplexes were processed with different efficiencies. Similar to bacterial and human system, the C-C heteroduplex was the weakest substrates. These results suggest that mouse cells possess a general, strand-specific mismatch repair system analogous to the Escherichia coli mutHLS and human mismatch repair system that contribute in a major way to the genetic stability. Finally, we established a reliable assay to examine the mismatch repair activity in mouse cells and this can be a tool to study the biochemical feature of mismatch repair in the future.

總目次…………………………………………………………I
圖目次…………………………………………………………III
表目次…………………………………………………………IV
摘要……………………………………………………………V
Abstract………………………………………………………VII
縮寫……………………………………………………………VIII
前言……………………………………………………………1
材料與方法……………………………………………………11
一、菌株………………………………………………………11
二、M12細胞株之繼代培養………………………………….11
三、f1MR1-PM載體之建構…………………………………..12
四、突變噬菌體f1PM mutant之建構……………………….14
五、f1PM系列雙股核酸及單股核酸之製備…………………16
六、具斷股之異雙股核酸製備………………………………18
七、超螺旋之異雙股核酸製備………………………………20
八、老鼠M12細胞核萃取液之製備………………………….21
九、異雙股核酸對測定用限制酵素之敏感度分析…………23
十、試管中之修復反應………………………………………25
十一、試管中修復情形之分析………………………………26
十二、M12細胞核萃取液中核酸內切酵素活性分析……….27
實驗結果………………………………………………………28
一、f1MR1-PM載體之建構…………………………………..28
二、各種噬菌體f1PM突變株之建構…………………………29
三、具斷股之異雙股核酸製備………………………………31
四、異雙股核酸對分析用限制酵素之敏感度分析…………33
五、試管中異雙股核酸之修復反應分析……………………35
六、M12細胞核萃取液中核酸內切酵素活性分析………….40
七、不同異雙股核酸在試管中修復反應之競爭實驗………41
八、試管中修復反應之需求性分析…………………………43
九、斷股位置對於試管中修復反應之影響…………………44
討論……………………………………………………………46
附圖……………………………………………………………51
附表……………………………………………………………65
參考文獻………………………………………………………70
圖目次
圖一、大腸桿菌甲基指引修復系統機制……………………51
圖二、真核細胞斷股指引修復系統機制……………………53
圖三、f1PM限制酵素位置圖及致突變處……………………54
圖四、f1PM突變噬菌體之選殖.…………………………….55
圖五、f1PM突變噬菌體核酸自動定序分析…………………56
圖六、具斷股之異雙股核酸製備流程………………………57
圖七、分析用限制酵素反應之專一性………………………58
圖八、異雙股核酸對分析用限制酵素反應之敏感度分析…59
圖九、修復反應溫度與修復效率之分析……………………60
圖十、修復反應中KCl濃度與修復效率之分析…………….61
圖十一、試管中修復反應之分析……………………………62
圖十二、M12細胞核萃取液中核酸內切酵素活性分析…….63
圖十三、斷股位置與修復效率之分析………………………64
表目次
表一、各種f1PM突變噬菌體之建構…………………………65
表二、各種f1PM異雙股核酸…………………………………66
表三、老鼠M12細胞萃取物斷股指引修復單一配對錯誤之效
率………………………………………………………67
表四、不同異雙股核酸在試管中修復反應之競爭實驗……68
表五、老鼠M12細胞核萃取液試管中修復反應之需求.……69

1.Modrich, P.: Mechanisms and biological effects of mismatch repair. Annu. Rev. Genet. 25:229-253, 1991.
2.Lahue, R. S., Au, K. G., and Modrich, P.: DNA mismatch correction in a defined system. Science 245:160-164, 1989.
3.Grilley, M., Griffith, J., and Modrich, P.: Bidirectional excision in methyl-directed mismatch repair. J. Biol. Chem. 268:11830-11837, 1993.
4.Lyons, S. M. and Schendel, P. F.: Kinetics of methylation in Escherichia coli K-12. J. Bacteriol. 159:421-423, 1984.
5.Lu, A. L., Clark, S. and Modrich, P.: Methyl-directed repair of DNA base-pair mismatches in vitro. Proc. Natl. Acad. Sci. USA 80:4639-4643, 1983.
6.Su, S. S. and Modrich, P.: Escherichia coli mutS-encoded protein binds to mismatched DNA base pairs. Proc Natl. Acad Sci. USA 83:5057-5061, 1986.
7.Grilley, M., Welsh, K. M., Su, S. S., and Modrich, P.: Isolation and characterization of the Escherichia coli mutL gene product. J. Biol. Chem. 264:1000-1004, 1989.
8.Au, K. G., Welsh, K. and Modrich, P.: Initiation of methyl-directed mismatch repair. J. Biol. Chem. 267: 12142-12148, 1992.
9.Cooper, D. L., Lahue, R. S. and Modrich, P.: Methyl-directed mismatch repair is bidirectional. J. Biol. Chem. 268:11823-11829, 1993.
10.Su, S. S., Lahue, R. S., Au, K. G., and Modrich, P.: Mispair specificity of methyl-directed DNA mismatch correction in vitro. J. Biol. Chem. 263:6829-6835, 1988.
11.Claverys, J. P. L., SA: Heteroduplex deoxyribonucleic acid base mismatch repair in bacteria. Microbiol. Rev. 50:133-165, 1986.
12.Dohet, C., Wagner, R. and Radman, M.: Methyl-directed repair of frameshift mutations in heteroduplex DNA. Proc. Natl. Acad. Sci. USA 83:3395-3397, 1986.
13.Fang, W. H., Wu, J. Y. and Su, M. J.: Methyl-directed repair of mismatched small heterologous sequences in cell extracts from Escherichia coli. J. Biol. Chem. 272:22714-22720, 1997.
14.Fishel, R. and Wilson, T.: MutS homologs in mammalian cells. Current Opinion in Genetics & Development. 7:105-113, 1997.
15.Johnson, R. E., Kovvali, G. K., Prakash, L. and Parkash, S.: Requirement of the yeast MSH3 and MSH6 genes for MSH2-dependent genomic stability. J. Biol. Chem. 271:7285-7288, 1996.
16.Karran, P.: Appropriate partners make good matches. Science 268:1857-1858, 1995.
17.Alani, E.: The Saccharomyces cerevisiae Msh2 and Msh6 proteins form a complex that specifically binds to duplex oligonucleotides containing mismatched DNA base pairs. Mol. Cell. Biol. 16:5604- 5615, 1996.
18.Habraken, Y., Sung, P., Prakash, L. and Prakash, S.: Binding of insertion/deletion DNA mismatches by the heterodimer of yeast mismatch repair proteins MSH2 and MSH3. Curr. Biol. 6:1185- 1187, 1996.
19.Marsischky, G. T., Filosi, N., Kane, M. F. and Kolodner, R. D.: Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair. Genes & Development 10: 407-420, 1996.
20.Reenan R., Kolodner R. D.: Characterization of insertion mutations in the Saccharomyces cerevisiae MSH1 and MSH2 genes: evidence for separate mitochondrial and nuclear functions. Genetics 132:975-985, 1992.
21.Chi N. W., Kolodner R. D.: Purification and characterization of MSH1, a yeast mitochondrial protein that binds to DNA mismatches. J. Biol. Chem. 269:29984-29992, 1994.
22.Ross-Macdonald P., Roeder G. S.: Mutation of a meiosis-specific MutS homolog decreases crossing over but not mismatch correction. Cell 79:1069-1080, 1994.
23.Hollingsworth N. M., Ponte L. and Halsey C.: MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair. Genes Dev. 9:1728-1739, 1995.
24.Hunter N., Borts R. H.: Mlh1 is unique among mismatch repair proteins in its ability to promote crossing-over during meiosis. Genes Dev. 11:1573-11582, 1997.
25.Habraken, Y., Sung, P., Prakash, L. and Prakash, S.: Enhancement of MSH2-MSH3-mediated mismatch recognition by the yeast MLH1-PMS1 complex. Curr. Biol. 7:790-793, 1997.
26.Acharya, S., Wilson, T., Gradia, S., Kane, M. F., Guerrette, S., Marsischky, G. T., Kolodner, R. D. and Fishel, R.: hMSH2 forms specific mispair-binding complexes with hMSH3 and hMSH6. Proc. Natl. Acad. Sci. USA 93:13629-13634, 1996.
27.Palombo, F., Iaccarino, I., Nakajima, E., Ikejima, M., Shimada, T. and Jiricny, J.: hMutSβ, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion loops in DNA. Curr. Biol. 6:1181-1184, 1996.
28.Umar, A., Risinger, J. I., Glaab, W. E., Tindall, K. R., Barrett, J. C. and Kunkel, T. A.: Functional overlap in mismatch repair by human MSH3 and MSH6. Genetics 148:1637-1646, 1998.
29.Szankasi P., Smith G. R.: A role for exonuclease I from S. pombe in mutation avoidance and mismatch correction. Science 267:1166-1169, 1995.
30.Tishkoff D. X., Boerger A. L., Bertrand P., Filosi N., Gaida G. M., Kane M. F., Kolodner R. D.: Identification and characterization of Saccharomyces cerevisiae EXOI, a gene encoding an exonuclease that interacts with MSH2. Proc. Natl. Acad. Sci. USA 94:7487-7492, 1997.
31.Fiorentini P., Huang K. N., Tishkoff D. X., Kolodner R. D. Symnigton L. S.,: Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro. Mol. Cell Biol. 17:2764-2773, 1997.
32.Tishkoff D. X., Amin N. S., Viars C. S., Arden K. C., Kolodner R. D.: Identification of a human gene encoding a homologue Saccharomyces cerevisiae EXOI, an exonuclease implicated in mismatch repair and recombination. Cancer Res. 58:5027-5031, 1998.
33.Schmutte C., Marinescu R. C., Sadoff M. M., Guerrette S., Overhauser J., Fishel R.: Human exonuclease I interacts with the mismatch repair protein hMSH2. Cancer Res. 58:4537-4542, 1998.
34.Johnson R. E., Gopala K. K., Prakash L., Prakash S.: Requirement of the yeast RTH1 5’to 3’ exonuclease for the stability simple repetitive DNA. Science 269:238-240, 1995.
35.Tishkoff D. X., Filosi N., Gaida G. M., Kolodner R. D.: A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair. Cell 88:253-263, 1997.
36.Tran HT., Gordenin DA., Resnick MA.: The 3’-5’ exonucleases of DNA polymerase delta and epsilon and the 5’-3’ exonuclease EXOI have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae.Mol. Cell Biol. 1999.
37.Gu L., Hong Y., McCulloch S., Watanabe H., Li G. M.: ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair. Nucleic acids Res. 26:1173-1178, 1998.
38.Umar A., Buermeyer A. B., Simon J. A., Thomas D. C., Clark A. B., Liskay R. M., Kunkel T. A.: Requirement for PCNA in DNA mismatch repair at a step preceding DNA synthesis. Cell 87:65-73, 1996.
39.Xie Y., Counter C., and Alani E.: Characterization of the repeat-tract instability and Mutator phenotypes conferred by a Tn3 insertion in RFC1, the large subunit of the yeast clamp loader. Genetics 1999.
40.Lin Y. L., Shivji M. K., Chen C., Kolodner R. D., Wood R. D., Dutta A: The evolutionarily conserved zinc finger motif in the largest subunit of human replication protein A is required for DNA replication and mismatch repair but not for nucleotide excision repair. J. Biol. Chem. 273:1453-1461, 1998.
41.Longley M. J., Pierce A. J. and Modrich P.: DNA polymerase delta is required for human mismatch repair in vitro. J. Biol. Chem. 272:10917-10921, 1997.
42.Fang, W. H. and Modrich, P.: Human strand-specific mismatch repair occurs by a bidirectional mechanism similar to that of the bacterial reaction. J. Biol. Chem. 268:11838-11844, 1993.
43.Modrich, P.: Strand-specific mismatch repair in mammalian cells. J. Biol. Chem. 272:24727-24730, 1997.
44.Umar, A., Boyer, J. C., and Kunkel, T. A.: DNA loop repair by human cell extracts. Science 266:814-816, 1994.
45.Macpherson P., Humbert O., and Karran P.: Frameshift mismatch recognition by the human MutS alpha complex. Mutat. Res. 408:55-66, 1995.
46.Liu, B., Parsons, R., Papadopoulos, N., Nicolaides, N. C., Lynch, H. T., Watson, P., Jass, J. R., Dunlop, M., Wyllie, A., Peltomaki, P., Chapelle, A., Hamilton, S. R., Vogelstein, B. and Kinzler, K.W.: Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nature medicine 2:169-172, 1996.
47.Papadopoulos, N., Nicolaides, N. C., Wei, Y. F., Ruben, S. M., Carter, K. C., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D., et al. Mutation of a mutL homolog in hereditary colon cancer. Science 263:1625-1629, 1994.
48.Shibata, D., Peinado, M. A., Ionov, Y., Malkhosyan, S., and Perucho, M.: Genomic instability in repeated sequences is an early somatic event in colorectal tumorigenesis that persists after transformation. Nat. Genet. 6:273-281, 1994.
49.Ionov, Y., Peinado, M. A., Malkhosyan, S., Shibata, D., and Perucho, M.: Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363:558-561, 1993.
50.Thibodeau, S. N., Bren, G., and Schaid, D.: Microsatellite instability in cancer of the proximal colon. Science 260:816-819, 1993.
51.Lindblom, A., Tannergard, P., Werelius, B., and Nordenskjold, M.: Genetic mapping of a second locus predisposing to hereditary non-polyposis colon cancer. Nat. Genet. 5:279-282, 1993.
52.Aaltonen, L. A., Peltomaki, P., Leach, F. S., Sistonen, P., Pylkkanen, L., Mecklin, J. P., Jarvinen, H., Powell, S. M., Jen, J. and Hamilton, S. R., et al: Clues to the pathogenesis of familial colorectal cancer. Science 260: 812-816, 1993.
53.Kolodner R. D.: Mismatch repair: mechanisms and relation to cancer susceptibility. Trends Biochem. Sci. 20:397-401, 1995.
54.Edelmann, W., Yang, K., Umar, A., Heyer, J., Lau, K., Fan, K., Liedtke, W., Cohen, P. E., Kane, M. F., et al: Mutation in the mismatch repair gene Msh6 causes cancer susceptibility. Cell 91:467-477, 1997.
55.Baker S. M., et al.: Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis. Cell 82:309-319, 1995.
56.Baker S. M., et al.: Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over. Nature Genet. 13:336-342, 1996.
57.Edelmann W., et al.: Meiotic pachytene arrest in MLH-1-deficient mice. Cell 85:1125-1134, 1996.
58.de Wind N., Dekker M., van Rossum A., van der Valk M.,te Riele H.: Mouse models for hereditary nonpolyposis colorectal cancer. Cancer Res. 58:248-255, 1998.
59.K. Jin Kim, Colette Kanellopoulos-langevin, Ruth M. Merwin, David H. Sachs, and Richard Asofsky: Establishment and characterization of BALB/c lymphoma lines with B cell properties. J. Immunology 122:549-554, 1979.
60.Jude Holmes, Jr., Susanna Clark, and Paul Modrich: Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines. Proc Natl. Acad Sci. USA 87:5837-5841, 1990.
61.Littman, S. J., Fang, W. H. and Modrich, P.: Repair of large insertiondeletion heterologies in human nuclear extracts is directed by a 5’ single-strand break and is independent of the mismatch repairsystem. J. Biol. Chem. 274:7474-7481, 1999.
62.Holliday, R.: A mechanism for gene conversion in fungi. Genetic Research 5:282-304, 1964.
63.Manivasakam, P., Rosenberg, S. M. and Hastings, P. J.: Poorly repaired mismatches in heteroduplex DNA are hyper-recombinagenic in Saccharomyces cerevisiae. Genetics 142:407-416, 1996.
64.Fishel, R. A., Siegel, E. C., and Kolodner, R. D.: Gene conversion in Escherichia coli. Resolution of heteroallelic mismatched nucleotides by co-repair. J. Mol. Biol. 188:147-157, 1986.
65.Dmitry O. Zharkov and Arthur P. Grollman: MutY DNA glycosylase: base release and intermediate complex formation. Biochemistry 37:12384-12394, 1998.
66.Arhonda Gogos, Jason Cillo, Neil D. Clarke, and A-Lien Lu: Specific recognition of A/G and A/7,8-dihydro-8-oxoguanine (8-oxoG) mismatches by Escherichia coli MutY : removal of the C-terminal domain preferentially affects A/8-oxoG recognition. Biochemistry 35:16665-16671, 1996.
67.Rayssiquier, R., D. S. Thaler, and M. Radman: The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature 342:396~401, 1989.
68.Su, S. S., Grilley, M., Thresher, R., Griffith, J., and Modrich, P.: Gap formation is associated with methyl-directed mismatch correction under conditions of restricted DNA synthesis. Genome 31:104-111, 1989.
69.Oda, O. Humbert, S. Fiumicino, M. Bignami, and P. Karran: Efficient repair of A/C mismatches in mouse cells deficient in long-patch mismatch repair. EMBO J. 19:1711-1718, 2000.