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

黃書毓(Shu-Yu Huang)

指導教授 ： 李財坤

國立臺灣大學/醫學院/微生物學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00759

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?琠w，並參與調控R-loop相關細胞功能，例如抗體變異性及DNA 複製。

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

並列摘要

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.

並列關鍵字

RNA/DNA hybrid ； R-loop ； AID ； DNA topoisomerase ； recombination ； RecF ； recBCsbcBC ； Mismatch repair

參考文獻

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