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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李財坤 | |
dc.contributor.author | Yi-Chun Chen | en |
dc.contributor.author | 陳苡均 | zh_TW |
dc.date.accessioned | 2021-06-15T16:14:28Z | - |
dc.date.available | 2020-09-25 | |
dc.date.copyright | 2015-09-25 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-08-17 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52425 | - |
dc.description.abstract | 伴隨轉錄作用的進行,去氧核醣核酸(DNA)產生拓樸(topology)結構的改變;其中,在核糖核酸聚合酶(RNA polymerase, RNAP)後方產生結構鬆散的負超螺旋(negative supercoils),使新生成的核糖核酸有機會和模板DNA配對,裸露非模板股的單股DNA,此結構稱為R-loop;裸露的單股DNA在B細胞的成熟過程中可以作為活化性胞嘧啶核苷脫氨酶(activation-induced cytidine deaminase, AID)的受質,作用在胞嘧啶,將其脫氨轉為尿嘧啶,經不同的修復重組途徑,產生體細胞超突變(somatic hypermutation, SHM)和免疫球蛋白類型轉換重組(class switch recombination, CSR)增加抗體歧異性(antibody diversification);然而,單股DNA結構較不穩定,容易斷裂或形成二級結構,且R-loop會阻礙RNAP行進,使轉錄中止,因此,R-loop在細胞中量的調控非常重要。已知細菌DNA拓樸異構酶III(topoisomerase III, TopB)具有解開負超螺旋的作用,解旋酶Q(RecQ)能消除DNA上的二級結構,且兩者亦能協同維持染色體穩定性。在這個論文中,我們以大腸桿菌作為模式生物,使用S9.6抗體偵測各細菌株之染色體中RNA-DNA雜交體(hybrid)的表現量,並在細菌中大量表現AID,以求得AID誘發突變倍數(AID-induced mutagenesis fold, ASM fold)來呈現R-loop多寡,用以探討TopB和RecQ在R-loop生成所扮演的角色;我們發現失去TopB功能會造成染色體中RNA-DNA雜交體表現量增加,再加上經過核糖核酸酶H(ribonuclease H, RNase H)處理後表現量降低,表示TopB能抑制R-loop生成;在細菌雙基因缺損株中自然突變率增加,但ASM fold卻大幅減少,可能因為此兩基因的缺陷已造成過多的生理功能缺陷,無法負荷由AID誘發產生的突變。在累積過多染色體損害而未修復的情況下,細菌會啟動SOS response,表現大量參與染色體修復的基因,並延遲細胞分裂,使細菌免於死亡,此反應會造成細胞型態的改變,產生絲狀生長(filamentation)的現象,所以也解釋我們發現TopB突變株有較高比例的絲狀生長現象,此現象也可能是R-loop增加所導致的結果,進一步研究是需要的。綜合以上結果,我們得知TopB藉由解開負超螺旋以抑制R-loop生成,在維持基因穩地性扮演重要的角色。 | zh_TW |
dc.description.abstract | During RNA transcription, the topology of DNA structure changes. Notably, loosely negative supercoiling formed behind RNA polymerase might allow the nascent RNA inserting into the negative supercoiled DNA helix and pairing with template DNA to form the RNA-DNA hybrid thus leading the formation of an abnormal R-loop structure consisting of RNA-DNA hybrid and single-strand DNA (ssDNA). The exposed ssDNA could be one potential targeting substrate of activation-induced cytidine deaminase (AID), which is activated in B cells and eventually leading to antibody diversification. AID acts on cytosine causing deamination and turning it into uracil. Through different repair and recombination pathways, the C ➔ U conversions further result into somatic hypermutation (SHM) and class switch recombination (CSR). In addition, the ssDNA region is also very unstable and prone to form secondary structures or DNA breakage. Furthermore, excessively accumulated R-loops impede the elongation of helical tracking enzymes on DNA to block transcription, replication and other DNA metabolism processes. For the reasons above, understanding of factors involved in the regulation of R-loop formation is important. Our lab’s previous results suggested that bacterial DNA topoisomerases, TopA and Gyrase, are regulatory factors of R-loop formation. Bacterial topoisomerase III, TopB, can also relax negatively supercoiled DNA and RecQ helicase can resolve secondary structures generated during transcription or replication processes. Moreover, TopB and RecQ could also act together to maintain genome integrity. Here, we used Escherichia coli as a model system and employed following assays to study the contributing factors. Specifically, S9.6 antibodies were used to detect the RNA-DNA hybrid in R-loop structure. Moreover, the AID-induced mutagenesis (ASM) assay with fluctuation test is also established by ectopically expressed AID into the wild-type and different mutant strains to obtain the ASM folds that indirectly represents the amount of R-loops. We observed that the signal intensity of S9.6 immuno-staining (i.e. the level of RNA-DNA hybrid) was elevated in the ΔtopB, ΔtopB ΔrecQ, but not ΔrecQ deleted cells. The level of S9.6 signaling was reduced only after RNase H treatment suggesting that S9.6 signaling represents R-loop and TopB can negatively regulate the formation of R-loop. Interestingly, spontaneous mutation frequency of ΔtopB ΔrecQ double mutant is two-fold higher than the wild-type strain but the ASM fold is decreased, possibly due to that the defects of double mutant cannot tolerate additional AID-stimulated mutagenesis and further leading to bacterial cell death. In addition, DNA damage activates the SOS response that subsequently leads to increased expression of repair and other factors and arrest of cell division (i.e. formation of filamentous cells). The percentage of abnormal filamentous cells increased in the ΔtopB mutant. These results are in agreement with the above observation and notion that the higher R-loop level in the ΔtopB mutant may lead to DNA damage and cellular filamentous phenotype. Taken together, TopB functions in inhibition of R-loop formation and plays an important roles in genome stability. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T16:14:28Z (GMT). No. of bitstreams: 1 ntu-104-R02445111-1.pdf: 4437833 bytes, checksum: 75c431bd942c630463313c2a7aaf6eed (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 誌謝…………………………………………………………………………………........i 中文摘要………………………………………………………………………………...ii ABSTRACT…………………………………………………………………………….iv INTRODUCTION……………………………………………………………………….1 1. DNA topoisomerases………………………………………………….………………1 1.1 Type I DNA topoisomerases………………………………………………….…..2 1.2 Type II DNA topoisomerases……………………………………………….…….2 1.3 The ability of DNA topoisomerase I and III in relaxation of negative supercoiling……………………………………………………………………….…..3 2. R-loop…………………………………………………………………………………4 2.1 Formation of R-loop………………………………………………………….…..4 2.2 Sequence requirements and structural characteristics of R-loop…………………5 2.3 Biological functions of R-loop……………………………………………….…..6 2.3.1 R-loop and cell growth………………………………………………………7 2.3.2 R-loop-mediated genome instability……………………………….………..7 2.4 Regulation of R-loop……………………………………………………………..8 2.5 Ribonucleotide misincorporation…………………………………………………9 3. RecQ helicases…………………………………………………………………….…10 4. Activation-induced cytidine deaminase (AID) and antibody diversification………..11 MATERIALS AND METHODS……………………………………………………….13 Escherichia coli strains and plasmids……………………………………….………13 Media and growth conditions……………………………………….………..……..13 Antibodies and reagents…………………………………………………….……….13 Dot blotting analysis……………………………………………………….…..……14 Preparation of competent cells…………………………………………….……..…15 Transformation…………………………………………………………….…..……15 AID-stimulated mutagenesis (ASM) assay…………………………………………15 Preparing cell lysates for Western blot analysis……………………………….……17 Western blot analysis……………………………………………………..…………17 Fluorescence microscopy……………………………………………………………18 Statistic analysis……………………………………………………..………………18 RESULTS………………………………………………………………………………19 TopB, but not RecQ, decreases the formation of RNA-DNA hybrid………….……19 The S9.6 signaling is reduced by RNase H treatment………………………..……..20 The AID-stimulated mutagenesis fold of ΔtopB ΔrecQ double mutant decreased, but spontaneous mutation frequency increased…………………..……………………..20 TopB and RecQ deficiency do not suffer plasmid-mediated lethality and AID-mediated growth defect ……………………………………………………………..21 The involvement of TopB and RecQ in the cellular filamentation………………….22 DISCUSSION………………………………………………………………………..…24 TopB functions in suppressing R-loop formation, but the ASM fold of CRL3 ΔtopB mutant did not increase.……………………………………………………………..24 Complementary of TopB may reverse the increased R-loop formation….…………24 The decrease of ASM fold in ΔtopB ΔrecQ mutantor strain………………………..24 Potential biological implications of and underlying mechanisms responsible for the phenotypic change of filamentous morphology…………………………………….25 REFERENCES..……………………………………………………………………..…27 TABLES AND FIGURES……………………………………………………..……….37 Table I. Escherichia coli strains used in this study.…………………………………37 Table II. Plasmids used in this study.………………………………………….…….38 Table III. Average mutation frequency of AID-stimulated mutagenesis (ASM) assay..…………………………………………………………………………….….39 Table IV. Colonies number on the rifampicin-containing plates after a time-course incubation……………………………………………………………………………40 Figure 1. TopB, but not RecQ, deficiency increases the cellular levels of RNA:DNA hybrid………………………………………………………………………………..41 Figure 2. RNase H treatment significantly reduced S9.6 signaling indicating the tight linking of RNA:DNA hybrid with R-loop.………………………………………….43 Figure 3. The fold of AID-stimulated mutagenesis (ASM) is lower in ΔtopB ΔrecQ double mutant.………………………………………………………………………44 Figure 4. No plasmid-mediated lethality and AID-mediated growth defect occurred in different strains………………………………………………………..………….47 Figure 5. Filamentous morphology changes in different mutants……………..……48 | |
dc.language.iso | en | |
dc.title | DNA拓樸異構酶III和RecQ解旋酶於調控R-loop⽣成所扮演的⾓色 | zh_TW |
dc.title | The role of TopB and RecQ in the regulation of R-loop formation | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧述諄,方偉宏 | |
dc.subject.keyword | 轉錄,R圈,DNA拓樸異構?,活化誘導胞嘧啶核?脫氨?,RecQ解旋?,轉錄相關重組,S9.6, | zh_TW |
dc.subject.keyword | RNA Transcription,R-loop,DNA Topoisomerases,Activation-induced Deaminase (AID),RecQ Helicases,Transcription-associated Recombination,S9.6, | en |
dc.relation.page | 49 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-08-18 | |
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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