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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李財坤(Tsai-Kun Li) | |
| dc.contributor.author | Shu-Yu Huang | en |
| dc.contributor.author | 黃書毓 | zh_TW |
| dc.date.accessioned | 2021-06-13T05:44:13Z | - |
| dc.date.available | 2006-08-03 | |
| dc.date.copyright | 2006-08-03 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-15 | |
| dc.identifier.citation | Adams, D.E., Shekhtman, E.M., Zechiedrich, E.L., Schmid, M.B. and Cozzarelli, N.R. (1992) The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication. Cell, 71, 277-288.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33653 | - |
| dc.description.abstract | 當核醣核酸聚合酶 (RNA polymerase) 在去氧核醣核酸 (DNA) 上進行轉錄(transcription) 並延長RNA時，有時會產生和RNA相關的結構，稱為R-loop。R-loop是由RNA和轉錄中的模板DNA (template DNA) 形成配對鍵結，而另一股DNA則是以單股的形式存在。R-loop的生理功能可以作為DNA複製 (replication) 時所必須的引子 (primer)，但是R-loop不正常的累積，可能會造成細胞存活率下降。近年來，發現造成抗體多異性 (antibody diversification) 的DNA基因重組 (recombination) 機制中轉錄所產生的R-loop扮演重要角色。免疫B細胞 (B cell) 中的蛋白，誘發活化性胞嘧啶核苷脫氨酶 (activation-induced cytidine deaminase, AID) 則參與在抗體發展更具高度辨識抗原的能力 (higher affinity)，和改變抗體的類型 (class switching)，藉此以因應不同的生理需求及對抗各類外來之病源。根據之前的文獻指出AID是作用在單股DNA，因此推論在R-loop 結構中的單股DNA可能是AID的作用目標受質。在本篇論文中，我們以細菌為操作模式系統，利用AID 刺激產生的基因突變 (AID-stimulated mutagenesis, ASM)為實驗方法，來探討DNA拓樸異構酶 (topoisomerases) 和參與基因重組的蛋白對R-loop形成的調控機制。本篇論文顯示，R-loop的確在AID發生基因重組過程中佔了很重要的地位。大腸桿菌中第一型DNA拓樸異構酶 (topoisomerase I, TopA)，第三型DNA拓樸異構酶 (topoisomerase III, TopB) 及第四型Topo IV拓樸異構酶 (topoisomerase IV, Topo IV) 會減少R-loop的形成而降低ASM; 相反地，第二型DNA拓樸異構酶 (gyrase) 則是增加R-loop和ASM的產生。另一方面，RecF 參與的重組途徑 (RecF pathway) 和錯配修復 (mismatch repair) 也在ASM中扮演重要的角色。本篇論文清楚指出拓樸異構酶利用調節DNA 拓樸 (Topology) 構型之能力來有效維持生物體內R-loop的恒定，並參與調控R-loop相關細胞功能，例如抗體變異性及DNA 複製。 | zh_TW |
| dc.description.abstract | The R-loop structure, an RNA/DNA hybrid with a single-stranded DNA region, has been observed during transcription elongation. Recently, the formation of transcription-associated R-loop and the activation-induced cytidine deaminase (AID) have been demonstrated to be tightly associated with the recombination events around antibody maturation. In addition to serve as recombination substrates, R-loop might also present as activation signal for cellular stress response and as primer for the initiation of DNA replication. In this thesis, we took advantage of a bacterial model system to investigate the roles of DNA topoisomerases and recombination enzymes in the regulation of R-loop formation which is indicated by changes in the level of the AID-stimulated mutagenesis (ASM). Our results provided the first evidence that R-loop is indeed the targeting substrates for AID-induced mutagenesis. In addition, R-loop is also responsible for plasmid-mediated lethality and cellular filamentations in recBCsbcBC mutant background. Consistent with the proposed role for TopA in suppression of R-loop formation, our results demonstrated that topA mutation and over-expression of TopA increased and reduced the frequencies of ASM, respectively. Similarly, deficiency of another Type I topoisomerase, TopB, also resulted in higher level of ASM. On the other hand, the presence of DNA gyrase increased ASM, possibly through its R-loop promoting activity. Interestingly, another Type II topoisomerase, Topo IV, plays a suppressing role in the function of R-loop. As for the recombination enzymes, enzymes in the mismatch repair (MMR) and RecF pathways were required for ASM. Furthermore, the RecF pathway, by modulation of R-loop formation, contributes to plasmid-mediated lethality and cellular filamentations in recBCsbcBC mutant background. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T05:44:13Z (GMT). No. of bitstreams: 1 ntu-95-R93445111-1.pdf: 5770052 bytes, checksum: 63fe03e229879b7c8f08d5a3053e9f1e (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 中文摘要
ABSTRACT INTRODUCTION…………………………………………………………………...1 1. R-loop 1-1. Formation of R-loop 1-2. Biological functions of R-loop 1-3. Regulation of R-loop 2. DNA topoisomerase 2-1. Type I enzymes: topoisomerase I (TopA) and topoisomerase III (TopB) in E. coli 2-2. Type II enzymes: gyrase and topoisomerase IV (Topo IV) in E. coli 3. Activation-induced cytidine deaminase (AID) and transcription for antibody diversification 3-1. Characteristics of AID: mechanisms of action and substrate requirements 3-2. Generation of antibody diversification following AID deamination 3-3. The assay of AID-stimulated mutagenesis (ASM) in E. coli 4. Recombination pathway in E. coli 4.1 RecBCD pathway 4.2 RecE pathway 4.3 RecF pathway 4.4 Mismatch repair (MMR) pathway SPECIFIC AIMS……………………………………………………………………14 MATERIAL AND METHODS……………………………………………………..15 -Escherichia coli strains -Plasmids -Media and growth conditions -Preparation of competent cells -Transformation -AID-stimulated mutagenesis assay -Plating efficiency -Fluorescence microscopy -DNA supercoiling analysis -Preparation for cell lysates for Western blot analysis -Western blot analysis RESULTS…………………………………………………………………….......... 21 -AID stimulates mutagenesis in a bacterial model system -Formation of R-loop is a prerequisite for AID-stimulated mutagenesis The roles of Type I topoisomerases in modulation of R-loop formation -DNA topoisomerase I (TopA) is as a negative regulator for the mutagenesis pathway induced by AID deamination 1. Mutation triggered by AID is enhanced by a deficiency of TopA in topA mutant 2. Over-expression of either TopA or RNaseH can efficiently reduce the folds of ASM in both wide type and topA mutant strains 3. R-loop associated negative supercoilings exist in topA mutant strain -DNA topoisomerase III (TopB) has the suppressing effect on AID-stimulated mutagenesis The roles of Type II topoisomerases in modulating R-loop formation -Gyrase is as a positive regulator contributing the higher frequency of ASM 1. Deficiency in gyrase activity decreases the frequency of ASM 2. Episomal expression of GyrB in gyrB20 (Ts) strain increases frequency of ASM. 3. Over-expression of either TopA or RNase H can still reduced the folds of ASM in gyrB mutant strain at semi-permissive temperature 4. Study of ASM in bacteria cells with double mutations in topA and gyrase genes revealed the differential abilities of TopA and gyrase in regulation of R-loop formation -DNA topoisomerase IV participated in AID-stimulated mutagenesis -The differential roles of recombination pathways in AID-stimulated recombination and other downstream events 1. The phenotype of recBCsbcBC mutant and its association with R-loop formation 2. The phenotypes of RecF pathway activation can suppress by TopA and RNaseH 3. Phenotypes relevant to the RecF pathway become more deleterious in recBCsbcBC mutant cells transformed the AID plasmid -Mismatch repair system is involved in the AID-stimulated mutagenesis DISCUSSIONS……………………………………………………………………..33 -The formation of R-loop versus the global and the local supercoiling -Through modulation of R-loop formation, DNA topoisomerase might contribute to antibody diversification -The role of RecF pathway in AID-stimulated mutegenesis -Other biological functions of R-loop REFERENCES………………………………………………………………...........39 FIGURES……………………………………………………………………...........47 -Table 1 Biochemical activities and cellular functions of E. coil proteins in this study -Table 2 List of E. coli strains used in this study -Figure 1 Formation of R-loop and its biological implications -Figure 2 Activation-induced deaminase (AID)-stimulated mutagenesis -Figure 3 R-loop structure presents a targeting substrate for AID and subsequently leading to AID-stimulated mutagenesis -Figure 4 TopA functions in the suppression of R-loop formation -Figure 5 Suppression of ASM by over-expression of TopA -Figure 6 TopA over-expression also suppressed the fold of ASM in topA mutant -Figure 7 Over-expression of RNase H also suppressed the fold of ASM in topA deletion or mutant cells -Figure 8 Plasmids extracted from topA deletion or mutant cells exhibit more negative supercoiled -Figure 9 The involvements of TopB and RecQ in regulation of R-loop formation -Figure 10 DNA gyrase promotes formation of R-loop -Figure 11 Over-expression of TopA or RNase H further suppressed the frequency of ASM in gyrase-deficient condition -Figure 12 Cells with double mutations in topA and gyrase genes revealed the differential abilities of TopA and gyrase in regulation of R-loop formation -Figure 13 The ASM of parER132C mutant cells (2822) were greater than that of wild type cells (1358) -Figure 14 AID contributed to plasmid-mediated lethality in various recombination-deficient backgrounds -Figure 15 Suppression of plasmid-mediated lethality by over-expression of either RNAse H or TopA in JC7623 (recBCsbcBC) cells -Figure 16 AID and RecF contributed to plasmid -mediated filamentous phenotype in recBCsbcBC backgrounds -Figure 17 R-loop contributes to the generation of cellular filamentation phenotype in JC7623 (recBCsbcBC) cells -Figure 18 The recombination pathways utilized by R-loop-associated mutagenesis -Figure 19 Mismatch repair (MMR) pathway is required for R-loop-associated mutagenesis -Figure 20 A schematic representation for the regulation of R-loop formation and R-loop-associated cellular responses | |
| dc.language.iso | en | |
| dc.subject | 拓樸異構酶 | zh_TW |
| dc.subject | 脫氨酶 | zh_TW |
| dc.subject | 誘發活化性胞嘧啶核苷 | zh_TW |
| dc.subject | 基因重組 | zh_TW |
| dc.subject | recombination | en |
| dc.subject | DNA topoisomerase | en |
| dc.subject | AID | en |
| dc.subject | RNA/DNA hybrid | en |
| dc.subject | R-loop | en |
| dc.subject | Mismatch repair | en |
| dc.subject | recBCsbcBC | en |
| dc.subject | RecF | en |
| dc.title | 探討細胞因子調控R-loop的形成及其生理意義:
AID造成之基因突變 | zh_TW |
| dc.title | Regulation of R-loop formation and its implications:
AID-stimulated mutagenesis | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧述諄,繆希椿 | |
| dc.subject.keyword | 拓樸異構酶,基因重組,誘發活化性胞嘧啶核苷,脫氨酶, | zh_TW |
| dc.subject.keyword | RNA/DNA hybrid,R-loop,AID,DNA topoisomerase,recombination,RecF,recBCsbcBC,Mismatch repair, | en |
| dc.relation.page | 73 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-17 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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