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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹迺立 | |
| dc.contributor.author | Tien-Jui Yen | en |
| dc.contributor.author | 顏天瑞 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:44:35Z | - |
| dc.date.copyright | 2009-09-15 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-03 | |
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Gadelle, Phylogenomics of DNA topoisomerases: their origin and putative roles in the emergence of modern organisms. Nucleic Acids Res, 2009. 37(3): p. 679-92. 43. Aubry, A., et al., First functional characterization of a singly expressed bacterial type II topoisomerase: the enzyme from Mycobacterium tuberculosis. Biochem Biophys Res Commun, 2006. 348(1): p. 158-65. 44. Jain, P. and V. Nagaraja, An atypical type II topoisomerase from Mycobacterium smegmatis with positive supercoiling activity. Mol Microbiol, 2005. 58(5): p. 1392-405. 45. Sioud, M., et al., Coumarin and quinolone action in archaebacteria: evidence for the presence of a DNA gyrase-like enzyme. J Bacteriol, 1988. 170(2): p. 946-53. 46. Forterre, P., et al., Origin and evolution of DNA topoisomerases. Biochimie, 2007. 89(4): p. 427-46. 47. Forterre, P., A. Bergerat, and P. Lopez-Garcia, The unique DNA topology and DNA topoisomerases of hyperthermophilic archaea. FEMS Microbiol Rev, 1996. 18(2-3): p. 237-48. 48. 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Cozzarelli, Formation and resolution of DNA catenanes by DNA gyrase. Cell, 1980. 20(1): p. 245-54. 55. Marini, J.C., K.G. Miller, and P.T. Englund, Decatenation of kinetoplast DNA by topoisomerases. J Biol Chem, 1980. 255(11): p. 4976-9. 56. Shapiro, T.A. and P.T. Englund, The structure and replication of kinetoplast DNA. Annu Rev Microbiol, 1995. 49: p. 117-43. 57. Sugino, A., N.P. Higgins, and N.R. Cozzarelli, DNA gyrase subunit stoichiometry and the covalent attachment of subunit A to DNA during DNA cleavage. Nucleic Acids Res, 1980. 8(17): p. 3865-74. 58. Baird, C.L., et al., Topoisomerase II drives DNA transport by hydrolyzing one ATP. Proc Natl Acad Sci U S A, 1999. 96(24): p. 13685-90. 59. Williams, N.L., A.J. Howells, and A. Maxwell, Locking the ATP-operated clamp of DNA gyrase: probing the mechanism of strand passage. J Mol Biol, 2001. 306(5): p. 969-84. 60. Osheroff, N., E.R. Shelton, and D.L. Brutlag, DNA topoisomerase II from Drosophila melanogaster. 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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23152 | - |
| dc.description.abstract | 當細胞進行DNA複製或轉錄時，DNA雙股的分離會造成DNA拓撲構型的改變，導致正超螺旋及負超螺旋的形成；若DNA雙股螺旋結構中累積的張力無法被移除，就會使細胞正常的生理功能無法繼續進行。除此之外，複製完成所產生彼此交聯的子染色體也必須先被分開，才能在細胞分裂時平均分配至子細胞中。在細菌體內，主要是藉由兩個高度同源卻功能互異的第IIA型DNA拓撲異構酶：DNA gyrase和topo IV，來解決上述DNA拓撲構型的問題。由於DNA gyrase具有引入負超螺旋的能力，其可迅速移除在DNA兩股分離處前方所產生的正超螺旋，並使細菌基因體處於浄負超螺旋的狀態，有助於啟動DNA複製及轉錄。相反的，topo IV則主要負責分離交聯的子染色體。對大多數細菌而言，這兩個酵素都是不可或缺的，二者共同維持細菌DNA結構的恆定。但全基因體序列分析卻發現少部分細菌體內只帶有一個第II型DNA拓撲異構酶，此單一第II型酵素是否具有特殊的活性，如同時帶有DNA gyrase和topo IV的功能，或只具有其中一者的功能即足以維持細菌正常生理功能，是本實驗所想要探討的方向。
在此我們藉由分析嗜高溫菌Aquifex aeolicus中單一第II型DNA拓撲異構酶(aeTopo II)的功能，來探討其如何維持細胞DNA拓撲結構的恆定。此酵素在序列同源性上較接近DNA gyrase，是由GyrA和GyrB兩個次單元所組成的四聚體。特別的是胺基酸序列比對的結果顯示其GyrA次單元之C端功能區(aeGyrA-CTD)和典型的GyrA-CTD有明顯差異。此功能區已知與gyrase引入負超螺旋的能力密切相關；然而aeGyrA-CTD序列長度卻明顯較短，且缺乏一個存在於所有gyrase中的保守性序列－GyrA box。這些差異都有可能導致aeTopo II的功能與典型的gyrase不同。 Aquifex aeolicus生長於極高溫的環境下，我們的結果亦顯示aeTopo II在85℃時才具有最好的活性。如同一般的DNA gyrase，當使用relaxed DNA做為反應受質時， aeTopo II可以將其轉變為負超螺旋的分子；但特別的是，此反應的進行並非完全仰賴ATP水解的能量。另一方面，在有ATP存在的情況下，aeTopo II可以催化負超螺旋DNA的relaxation。相較於E. coli gyrase需要ATP才能引入負超螺旋，而ATP的存在反而會使其DNA relaxation的活性消失，aeTopo II的活性及其與ATP的相互關係就顯得很特別。除此之外，aeTopo II能像topo IV一般，將kDNA network有效的分離開來。綜合以上結果可知，隨著使用的受質不同，aeTopo II能呈現相對的(-) supercoiling或relaxation activity，而在ATP存在時反應速率皆會加快。我們亦發現移除aeGyrA-CTD只會減緩反應的進行，而不影響其對不同受質的催化反應，表示此C端功能區的存在與否對aeTopo II之活性變化並沒有決定性的角色。 由於在高溫環境下DNA melting的效應，使得原本relaxed DNA分子上會產生互補的正超螺旋；這些儲存於高溫所產生的DNA超螺旋中的能量似乎可以直接被aeTopo II利用而進行催化反應，而所觀察到的ATP-independent supercoiling activity可能即為aeTopo II對高溫下產生的正超螺旋進行relaxation的結果。因此aeTopo II是一個具有relaxation活性，而不具有如同E. coli gyrase之主動引入(-) suerpcoils的活性。另一方面，由於ATP的存在會進一步加速反應的進行，推測ATP結合所造成的構型改變有助其捕捉另一DNA片段而完成催化反應。目前已知，高溫菌中具有一種特化的第I型拓撲異構酶reverse gyrase，此酵素能主動引入正超螺旋，以避免過度的strand unwinding。鑑於高溫下正超螺旋的重要，因此aeTopo II不具主動引入負超螺旋活性應屬合理，而aeTopo II可以再進一步藉由relaxation，與reverse gyrase共同維持高溫菌中genomic DNA超螺旋結構的恆定。 由於aeTopo II同時具有relaxation和decatenation的活性，其單獨存在就足以維持細胞內複製、轉錄的正常進行。從演化的角度來看，由於Aquifex aeolicus是最早從古細菌中演化而來的真細菌之一，而aeTopo II又主要是進行DNA relaxation，因此推論早期細菌所帶有的單一第II型DNA拓撲異構酶主要是具有DNA relaxation 與decatenation activities，之後因應不同菌種的生理需求，才再演化出具有supercoiling activity的DNA gyrase。我們的結果亦顯示單純從序列上的同源性來分類第II型DNA拓撲異構酶是有其限制之處；唯有對更多不同細菌的第II型DNA拓撲異構酶進行功能分析，才能使我們更加了解每一個第II型DNA拓撲異構酶是如何執行其功能以維持不同細菌的生理需求。 | zh_TW |
| dc.description.abstract | The intertwined structure of duplex DNA is known to cause topological problems during DNA transactions, in which processive strand unwinding generates the under- and over-winding on the flanking duplex regions. If not removed, the accumulation of superhelical strains will halt DNA duplication and gene expression. In addition, newly replicated daughter chromosomes are interlinked and must be segregated prior to cell division. In bacteria, two closely related yet functionally distinct type IIA topoisomerases (topos), DNA gyrase and topo IV, are responsible for resolving these topological problems. With the unique (-) supercoiling activity, gyrase removes the (+) supercoils in front of the replication forks and maintains bacterial genomes in a slightly underwound state, promoting the initiation of transcription and replication. In contrast, topo IV is specialized for decatenation of tangled daughter chromosomes. For most bacteria, these two enzymes are both essential and work in concert to maintain the superhelical homeostasis. However, a few eubacteria possess only one type II topo. It remains controversial whether this lone type II topo functions mainly as a gyrase or a topo IV, or if it possesses both the (-) supercoiling and decatenation activities, to sustain cell viability.
Here we characterized the lone type II topo encoded by the genome of Aquifex aeolicus, a hyperthermophilic eubacterium. This enzyme is currently annotated as a gyrase, composed of the GyrB and GyrA subunits. However, sequence analysis reveals that the C-terminal domain of A. aeolicus GyrA (aeGyrA-CTD), a domain known to confer the (-) supercoiling activity of gyrase, is very different from other GyrA-CTD. Specifically, the aeGyrA-CTD is significantly shorter in length and lacks a characteristic GyrA box (QRRGGKG) found in all gyrases. These differences may contribute to its unique activity as the sole type II topo. The A. aeolicus topo II was purified to homogeneity, whose optimal activity was observed at around 85℃, consistent with the extremely high growing temperature of this bacterium. At 85℃ A. aeolicus topo II was found to introduce (-) supercoils into relaxed DNA molecules, indicating that it may function as a gyrase. Surprisingly, while other gyrases require ATP to drive this reaction, A. aeolicus topo II is not strictly ATP-dependent. Furthermore, when the (-) supercoiled DNA was incubated with A. aeolicus topo II, it becomes more relaxed even in the presence of ATP. These properties are distinct from those of E. coli gyrase, whose (-) supercoiling activity requires ATP and it does not exhibit relaxation activity in the presence of ATP. In addition, A. aeolicus topo II efficiently resolves interlinked kDNA network, suggesting that it may act as a topo IV-like decatenase in vivo. Therefore, A. aeolicus topo II is unique in that, depending on substrates, either (-) supercoiling or relaxation activity can be observed, with both activities being accelerated by ATP hydrolysis. Moreover, removal of aeGyrA-CTD merely reduces the reaction rate but the catalytic pattern of A. aeolicus topo II on different substrates was not affected, indicating that this domain does not play a dominant role in mediating the holoenzyme activity. The unique ATP-independent supercoiling activity at high temperature can be explained by relaxation of (+) supercoils. Due to the emergence of compensatory (+) supercoils on relaxed closed circular DNA associated with DNA unwinding at high temperature, A. aeolicus topo II releases the free energy stored as (+) supercoils to achieve DNA relaxation. Therefore, it appears that A. aeolicus topo II possesses merely relaxation and no (-) supercoiling activity, and the relaxation process is further facilitated by ATP hydrolysis, where the nucleotide binding-induced structural changes leads to more efficient capture of a second DNA segment for strand passage. The biased activity toward DNA relaxation of A. aeolicus topo II seems reasonable with the concomitant presence of the hyperthermophilic-specific reverse gyrase in vivo. Reverse gyrase actively generates (+) supercoils to overcome DNA denaturation, wheras A. aeolicus topo II functions as a decatenase to support chromosome segregation. In addition, this enzyme also relaxes excessive superhelical strains to maintain DNA superhelical homeostasis. By harboring efficient relaxation and decatenation activities, A. aeolicus topo II is functionally more related to bacterial topo IV. Being one of the earliest diverging eubacteria, our result implicates that the relaxation/decatenation activity evolved earlier in the prototype of topo II, and (-) supercoiling activity may not be required for the growth of ancient bacteria. Further studies on other type II topos from a wide range of bacteria will provide more information regarding how the topoisomerase activity can be fine-tuned to meet specific physiological requirements. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:44:35Z (GMT). No. of bitstreams: 1 ntu-98-R96442016-1.pdf: 2667625 bytes, checksum: c2ebbb569de5f9e30bf8d4b4c9a5e2b8 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 中文摘要 I
Abstract IV Introduction 1 1. DNA topology 1 DNA structure and DNA supercoiling 1 Roles of DNA supercoiling in DNA metabolism 2 2. DNA topoisomerases 3 Type II DNA topoisomerases 3 General quaternary structure of type IIA topos 4 Proposed catalytic mechanism of type IIA topos 4 Two speciallized type IIA topos collaborate to maintain chromosome topology in bacteria 5 3. The C-terminal domain of the A subunit in bacterial type IIA topos mediates T-segment selectivity and the directionality of supercoiling 6 Function and structure of bacterial type IIA topo CTDs 6 The GyrA box is required for the supercoiling reaction of gyrase 8 4. The role of single type IIA topoisomerase in certain microorganisms 8 5. Evolution of prokaryotic type II DNA topoisomerases 10 Specific aims 12 Figures 14 Fig. I. Geometrical models of DNA crossings. 14 Fig. II. Topological problems associated with DNA transactions. 15 Fig. III. Proposed quaternary structure and catalytic mechanism of type IIA topoisomerases. 16 Fig. IV. The CTDs of bacterial type IIA topoisomerases controls holoenzyme activity. 17 Fig. V. Multiple sequence alignment of the A subunit of prokaryotic type IIA topo.. 18 Materials and Methods 20 Cloning 20 Protein overexpression and purification 20 - GyrA, GyrB, GyrA-NTD, GyrA-CTD, and wild type/CTD-truncated holoenzyme complex from A. aeolicus 20 - Preparation of selenomethionyl aeGyrA-CTD 21 Crystallization of aeGyrA-CTD 21 DNA supercoiling assay 22 DNA relaxation assay 23 Decatenation assay 23 DNA ligase readout assay 24 Results 25 Purification of A. aeolicus topo II 25 Crystallization and data collection of A. aeolicus GyrA-CTD (aeGyrA-CTD) 25 Functional characterization 26 - ATP-independent negative supercoiling activity at high temperature 26 - Weak ATP-independent relaxation activity at high temperature 27 - Efficient decatenation activity on kinetoplast DNA 29 Conclusion 30 Discussion 31 A. aeolicus topo II relaxes (+) DNA supercoils generated at high temperature 31 The role of ATP on the catalysis of A. aeolicus topo II 33 The contribution of aeGyrA-CTD to the catalytic kinetics 35 The biological significance of the lone type II DNA topoisomerase 36 Figures 39 Fig. 1. Purification and crystallization of individual subunit, domain as well as the reconstituted holocomplex of A. aeolicus topo II (aeTopo II). 39 Fig. 2. Analysis of the functional interaction between GyrA/GyrA-NTD and GyrB by gel filtration column. 40 Fig 3. Comparison between the activity of E. coli DNA gyrase and A. aeolicus topo II on different substrates at ambient temperature. 41 Fig 4. Negative supercoiling activity of wild type and CTD-truncated A. aeolicus topo II. 42 Fig. 5. DNA relaxation activity of wild type and CTD-truncated A. aeolicus DNA topo II. 44 Fig. 6. The decatenation activity of A. aeolicus topo II. 45 Fig. 7. Decatenation assay of wild type and CTD-truncated B. stearothermophilus topoisomerase IV. 46 Fig. 8. Ligase readout for alteration of DNA superhelicity along with temperature variation. 47 References 48 | |
| dc.language.iso | en | |
| dc.subject | 嗜高溫菌 | zh_TW |
| dc.subject | 第II型DNA拓撲異構酶 | zh_TW |
| dc.subject | Hyperthermophile | en |
| dc.subject | GyrA-CTD | en |
| dc.subject | A. aeolicus | en |
| dc.subject | Type II DNA topoisomerase | en |
| dc.subject | DNA gyrase | en |
| dc.title | 嗜高溫菌Aquifex aeolicus之單一第II型DNA拓撲異構酶的功能及結構分析 | zh_TW |
| dc.title | Functional and structural characterization of the lone type II DNA topoisomerase from the hyperthermophilic bacterium Aquifex aeolicus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李財坤,張功耀 | |
| dc.subject.keyword | 第II型DNA拓撲異構酶,嗜高溫菌, | zh_TW |
| dc.subject.keyword | Type II DNA topoisomerase,DNA gyrase,Hyperthermophile,A. aeolicus,GyrA-CTD, | en |
| dc.relation.page | 52 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2009-08-03 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-98-1.pdf 未授權公開取用 | 2.61 MB | Adobe PDF |
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