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標題: | 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 Regulation of R-loop formation and its implications: AID-stimulated mutagenesis |
作者: | Shu-Yu Huang 黃書毓 |
指導教授: | 李財坤(Tsai-Kun Li) |
關鍵字: | 拓樸異構酶,基因重組,誘發活化性胞嘧啶核苷,脫氨酶, RNA/DNA hybrid,R-loop,AID,DNA topoisomerase,recombination,RecF,recBCsbcBC,Mismatch repair, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 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。 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33653 |
全文授權: | 有償授權 |
顯示於系所單位: | 微生物學科所 |
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