細胞分裂過程中DNA的複製與分配是一項很重要的步驟。Par(partition)即是參與此重要功能的系統，其使複製完成的染色體與質體準確地移動到子細胞內確保遺傳物質數量皆相等。Par系統包含Spo0J及Soj兩種蛋白質與類著絲粒的parS DNA序列。於幽門螺旋桿菌的Par系統中為HpSpo0J與HpSoj兩種蛋白質。Soj是個ATPase。Spo0J是個DNA結合蛋白質會與parS DNA序列結合並且提高Soj水解ATP的速率。利用電泳遷移率變動分析(EMSA)得知 parS DNA (15和17bp)序列與HpSpo0J結合。同樣地運用電泳遷移率變動分析得知55bp的parS DNA與HpSpo0J及HpSoj蛋白質的結合。透過鉬藍試驗(molybdenum-blue assay)得知HpSoj水解ATP速率為0.96 ± 0.3 mole Pi/ mole of Soj．hr。此外,HpSpo0J或parS-55bp個別的參與均會提高HpSoj水解ATP的速率。當HpSpo0J與parS-55bp同時加入時，HpSoj的ATP水解速率提高了45倍。由此猜測HpSpo0J HpSoj parS-55bp三者之間或許會有交互作用。HpSpo0J-HpSoj- parS DNA複合物晶體的培養尚未成功。推測可能原因為HpSpo0J的不穩定與parS-55bp DNA序列太長。

DNA replication and partition played an important role in cell division. To ensure the amounts of genetic material in daughter cells are equal, Par(partition) system makes the replicated DNA moves to daughter cells precisely. Par system contains Spo0J, Soj and centromere-like parS DNA sequence. HpSpo0J and HpSoj are the two proteins from H.pylori for Par system. Soj is an ATPase and binding to nonspecific DNA sequence. Spo0J is a DNA binding protein for parS DNA binding specifically and it can stimulate the ATP hydrolysis of Soj. Using electrophoretic mobility shift assay to detect the parS DNA (15 and 17bp) binding to HpSpo0J. Also, a 55bp parS DNA was used to study the DNA binding to HpSpo0J and HpSoj, respectively by EMSA. The ATPase activity of HpSoj was performed by the molybdenum-blue assay and the ATPase activity of 0.96 ± 0.3 mole Pi/ mole of Soj．hr was determined. HpSpo0J or parS-55bp DNA will stimulate the ATPase activity. The additional of HpSpo0J and parS-55bp DNA mixed the ATPase activity is increased 45 folds the ATPase activity of HpSoj. We speculate that HpSpo0J, HpSoj and parS-55bp may work corporately. Unfortunately, the crystallization of the protein-nucleotide complex, the Spo0J-Soj-parS DNA complex was fail. The fail of crystallization might because the instability of HpSpo0J and the long length of parS-55bp.

中文摘要 I
Abstract II
謝誌 III
內容 IV
第一章 前言 1
1.1幽門螺旋桿菌(Helicobacter pylori) 1
1.2細菌DNA分配系統(partition system) 2
1.3 Par ABS 系統 3
1.4 Spo0J 4
1.5 Soj 5
第二章 實驗材料與方法 6
2.1 HpSpo0J與HpSpo0JN240基因選殖 6
2.2 HpSoj基因選殖 6
2.3蛋白質製備與純化 7
2.4 HpSpo0J-parS複合物製備 7
2.5粒徑篩析層析(size exclusion chromatography) 8
2.6電泳遷移率變動分析(electrophoretic mobility shift assay) 8
2.7 ATPase活性分析 9
2.8蛋白質晶體培養 9
第三章 實驗結果 11
3.1 HpSpo0J多序列比對 11
3.2 HpSoj多序列比對 12
3.3蛋白質製備、純化與特徵 12
3.3.1 HpSpo0J 12
3.3.2 HpSpo0J截短蛋白質 13
3.3.3 HpSoj 13
3.4 HpSpo0J的DNA結合能力分析 14
3.5 HpSoj、HpSpo0J與parS-55bp的結合能力分析 14
3.6 HpSoj ATPase活性分析 15
3.7 HpSoj、HpSpo0JN240與parS-55bp的結合能力分析 16
3.8蛋白質晶體培養 16
第四章 總結 18
第五章 實驗數據及圖表 20
第六章 參考文獻 35

1. Marshall, B.J. and J.R. Warren, Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet, 1984. 1(8390): p. 1311-5.
2. Goodwin, C.S. and J.A. Armstrong, Microbiological aspects of Helicobacter pylori (Campylobacter pylori). Eur J Clin Microbiol Infect Dis, 1990. 9(1): p. 1-13.
3. Danesh, J., Helicobacter pylori and gastric cancer: time for mega-trials? Br J Cancer, 1999. 80(7): p. 927-9.
4. Shibayama, K., et al., A novel apoptosis-inducing protein from Helicobacter pylori. Mol Microbiol, 2003. 47(2): p. 443-51.
5. Dunn, B.E., H. Cohen, and M.J. Blaser, Helicobacter pylori. Clin Microbiol Rev, 1997. 10(4): p. 720-41.
6. Kuck, D., et al., Vacuolating cytotoxin of Helicobacter pylori induces apoptosis in the human gastric epithelial cell line AGS. Infect Immun, 2001. 69(8): p. 5080-7.
7. Blaser, M.J., et al., Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res, 1995. 55(10): p. 2111-5.
8. Tomb, J.F., et al., The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature, 1997. 388(6642): p. 539-47.
9. Hatakeyama, M., Helicobacter pylori and gastric carcinogenesis. J Gastroenterol, 2009. 44(4): p. 239-48.
10. Fraser, C.M., et al., The minimal gene complement of Mycoplasma genitalium. Science, 1995. 270(5235): p. 397-403.
11. Walczak, C.E., S. Cai, and A. Khodjakov, Mechanisms of chromosome behaviour during mitosis. Nat Rev Mol Cell Biol, 2010. 11(2): p. 91-102.
12. Ebersbach, G. and K. Gerdes, Plasmid segregation mechanisms. Annu Rev Genet, 2005. 39: p. 453-79.
13. Ogura, T. and S. Hiraga, Partition mechanism of F plasmid: two plasmid gene-encoded products and a cis-acting region are involved in partition. Cell, 1983. 32(2): p. 351-60.
14. Yamaichi, Y. and H. Niki, Active segregation by the Bacillus subtilis partitioning system in Escherichia coli. Proc Natl Acad Sci U S A, 2000. 97(26): p. 14656-61.
15. Salje, J., P. Gayathri, and J. Lowe, The ParMRC system: molecular mechanisms of plasmid segregation by actin-like filaments. Nat Rev Microbiol, 2010. 8(10): p. 683-92.
16. Gerdes, K., J. Moller-Jensen, and R. Bugge Jensen, Plasmid and chromosome partitioning: surprises from phylogeny. Mol Microbiol, 2000. 37(3): p. 455-66.
17. Davis, M.A., K.A. Martin, and S.J. Austin, Biochemical activities of the parA partition protein of the P1 plasmid. Mol Microbiol, 1992. 6(9): p. 1141-7.
18. Davey, M.J. and B.E. Funnell, Modulation of the P1 plasmid partition protein ParA by ATP, ADP, and P1 ParB. J Biol Chem, 1997. 272(24): p. 15286-92.
19. Hayes, F., et al., The homologous operons for P1 and P7 plasmid partition are autoregulated from dissimilar operator sites. Mol Microbiol, 1994. 11(2): p. 249-60.
20. Lee, M.J., et al., Characterization of the Soj/Spo0J chromosome segregation proteins and identification of putative parS sequences in Helicobacter pylori. Biochem Biophys Res Commun, 2006. 342(3): p. 744-50.
21. Lin, D.C. and A.D. Grossman, Identification and characterization of a bacterial chromosome partitioning site. Cell, 1998. 92(5): p. 675-85.
22. Godfrin-Estevenon, A.M., F. Pasta, and D. Lane, The parAB gene products of Pseudomonas putida exhibit partition activity in both P. putida and Escherichia coli. Mol Microbiol, 2002. 43(1): p. 39-49.
23. Nardmann, J. and W. Messer, Identification and characterization of the dnaA upstream region of Thermus thermophilus. Gene, 2000. 261(2): p. 299-303.
24. Ireton, K., N.W.t. Gunther, and A.D. Grossman, spo0J is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subtilis. J Bacteriol, 1994. 176(17): p. 5320-9.
25. Montecucco, F., et al., Inflammation in the pathophysiology of essential hypertension. J Nephrol, 2011. 24(1): p. 23-34.
26. Breier, A.M. and A.D. Grossman, Whole-genome analysis of the chromosome partitioning and sporulation protein Spo0J (ParB) reveals spreading and origin-distal sites on the Bacillus subtilis chromosome. Mol Microbiol, 2007. 64(3): p. 703-18.
27. Leonard, T.A., P.J. Butler, and J. Lowe, Structural analysis of the chromosome segregation protein Spo0J from Thermus thermophilus. Mol Microbiol, 2004. 53(2): p. 419-32.
28. Autret, S., R. Nair, and J. Errington, Genetic analysis of the chromosome segregation protein Spo0J of Bacillus subtilis: evidence for separate domains involved in DNA binding and interactions with Soj protein. Mol Microbiol, 2001. 41(3): p. 743-55.
29. Lobocka, M. and M. Yarmolinsky, P1 plasmid partition: a mutational analysis of ParB. J Mol Biol, 1996. 259(3): p. 366-82.
30. Gruber, S. and J. Errington, Recruitment of condensin to replication origin regions by ParB/SpoOJ promotes chromosome segregation in B. subtilis. Cell, 2009. 137(4): p. 685-96.
31. Figge, R.M., J. Easter, and J.W. Gober, Productive interaction between the chromosome partitioning proteins, ParA and ParB, is required for the progression of the cell cycle in Caulobacter crescentus. Mol Microbiol, 2003. 47(5): p. 1225-37.
32. Scholefield, G., et al., Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation. Mol Microbiol, 2011. 79(4): p. 1089-100.
33. Schumacher, M.A. and B.E. Funnell, Structures of ParB bound to DNA reveal mechanism of partition complex formation. Nature, 2005. 438(7067): p. 516-9.
34. Schumacher, M.A., K.M. Piro, and W. Xu, Insight into F plasmid DNA segregation revealed by structures of SopB and SopB-DNA complexes. Nucleic Acids Res, 2010. 38(13): p. 4514-26.
35. Leonard, T.A., P.J. Butler, and J. Lowe, Bacterial chromosome segregation: structure and DNA binding of the Soj dimer--a conserved biological switch. Embo j, 2005. 24(2): p. 270-82.
36. Murray, H. and J. Errington, Dynamic control of the DNA replication initiation protein DnaA by Soj/ParA. Cell, 2008. 135(1): p. 74-84.
37. Quisel, J.D., D.C. Lin, and A.D. Grossman, Control of development by altered localization of a transcription factor in B. subtilis. Mol Cell, 1999. 4(5): p. 665-72.
38. Dunham, T.D., et al., Structural basis for ADP-mediated transcriptional regulation by P1 and P7 ParA. Embo j, 2009. 28(12): p. 1792-802.
39. Bartolommei, G., M.R. Moncelli, and F. Tadini-Buoninsegni, A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases). PLoS One, 2013. 8(3): p. e58615.
40. Puhar, A., et al., Comparison of the pH-induced conformational change of different clostridial neurotoxins. Biochem Biophys Res Commun, 2004. 319(1): p. 66-71.
41. Koonin, E.V., A superfamily of ATPases with diverse functions containing either classical or deviant ATP-binding motif. J Mol Biol, 1993. 229(4): p. 1165-74.
42. Hester, C.M. and J. Lutkenhaus, Soj (ParA) DNA binding is mediated by conserved arginines and is essential for plasmid segregation. Proc Natl Acad Sci U S A, 2007. 104(51): p. 20326-31.