探討轉錄所伴隨生成的R-loop之機制與調控

The Regulation of Transcription-associated R-loop Formation

梁云馨(Yun-Hsin Liang)

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(2011年)

https://doi.org/10.6342/NTU.2011.03299

摘要

去氧核醣核酸(DNA)進行轉錄時，座落於核醣核酸聚合酶(RNA polymerase)之後新轉錄出的核醣核酸(RNA)有時會與模板去氧核醣核酸進行配對互補，並暴露出非轉錄的單股DNA (single-stranded DNA, ssDNA)，這樣的結構稱為R-loop。近年來研究發現，免疫B細胞成熟過程中，誘發活化性胞嘧啶核苷脫氨酶(Activation-induced cytidine deaminase, AID)會對ssDNA作用，促進胞嘧啶脫氨轉化成尿嘧啶，進而產生突變，而造成抗體產生多異性(antibody diversification)；因此，我們與其他實驗室認為R-loop提供了ssDNA作為AID受質。此外，R-loop也作為DNA複製(replication)時的引子(primer)，然而，過度累積的R-loop也會不利於細胞存活。目前在細胞中R-loop的生成及調控機制都尚未被釐清；我們利用大腸桿菌做為操作模式系統，利用在細菌中大量表現AID來刺激產生突變。藉由在不同細菌株中產生不同的AID刺激突變倍數(AID-stimulated mutagenesis fold, ASM fold)來表示所產生R-loop的程度，以此來鑑別參與在其中的細胞因子。此篇論文中，我們探討類核體相關蛋白質(Nucleoid-associated proteins, NAPs)是否參與在R-loop的形成和調控，我們發現Fis缺失的菌種其ASM倍數有降低的現象。此外，我們實驗室先前發現，RecF活化的菌株(RecF-activated strain, JC7623)有極高的ASM倍數，除此之外，RecF活化的菌株也擁有許多獨特的顯型，例如：質體造成的死亡(plasmid-mediated lethality)、滾環狀(rolling-circle)複製以及細菌絲狀生長(cellular filamentation)；而這些顯型都可以被額外表現的第一型拓譜異構酶(TopA)或核糖核酸酶H (RNaseH)所抑制。因此，我們認為RecF在R-loop的形成中，可能扮演了重要的角色；而為了得知R-loop調控是由RecF蛋白質或是RecF途徑來參與，我們利用RecF活化JC7623細菌額外突變了RecA的菌株(JC7940)來觀察這些顯型是否存在。我們研究結果顯示，額外突變了RecA也無法降低刺激突變倍數以及減少細菌絲狀生長的現象；然而，可以稍微減緩質體造成的死亡。綜合以上結果，我們認為RecF蛋白質本身對於促使R-loop的增加扮演主要的角色。此暗示了R-loop在細胞中的重要性，也找出調控此結構的分子，此有利於更進一步了解免疫細胞成熟過程中的程序與步驟。

關鍵字

轉錄 ； DNA拓樸異構酶活化誘導胞嘧啶核苷脫氨酶R圈 ； 基因重組 ； RecF途徑 ； RecA 蛋白質 ； 類核體相關蛋白質

並列摘要

During transcription elongation, the nascent RNA transiently anneals with the DNA template strand behind the RNA polymerase. This RNA-DNA hybrid with a single-stranded DNA (ssDNA) region is called R-loop. Our recent results and other reports have suggested that, R-loop might serve as the substrate for activation-induced deaminase (AID) during B cell maturation and antibody dicersification. In addition, R-loop has also been suggested to function as the primer for initiation of DNA replication. Furthermore, excess amount of R-loop formed in cells might be harmful. However, the regulation of R-loop formation remains largely unclear. Here, we sought to identify cellular factors that regulate the formation of R-loop by taking advantage of the AID-stimulated mutagenesis (ASM) assay in the bacterial model system. The ASM assay was employed to further explore the contributing factors involved in R-loop formation. Our data suggested that nucleoid-associated protein Fis might be involved in the negative regulation of R-loop formation Our previous studies have shown that the ASM level was much higher in the RecF-activated strain (JC7623, recBC sbcBC) than that in the wild type strain. Moreover, the activated RecF pathway contributed to the plasmid-mediated lethality, run-away plasmid replication and cellular filamentation phenotype. Expression of AID further severed these phenotypes. All of above phenotypes could be suppressed by additional expression of functional RNase H or TopA, two identified suppressors for R-loop formation. We thus suggested that RecF may play a role in the R-loop formation rather than in the downstream recombination pathway leading to mutagenesis. Here we investigated whether and how RecF pathway or RecF itself are involved in the R-loop formation and regulation. In this regard, we examined the RecF-associated phenotype in the RecF-activated strain with additional recA mutation (JC7940, recBC sbcBC recA). Our results showed that additional RecA mutation in RecF-activated strain JC7940 cannot reduce the ASM fold, suggesting that the RecA-independence of AID-mediated recombination. The cellular filamentation is also evident in JC7940 cells. Although additional RecA mutation in JC7940 could diminish the vector-mediated lethality, the additional RecA mutation could only partially reduce the plasmid-mediated lethality in JC7940 transform with AID-expressing plasmid. We thus suggest that RecF itself plays the major role in the R-loop formation. In vitro R-loop formation assay with purified proteins were under development to provide further supports for above hypothesis. In this regard, we have successfully expressed and purified several bacterial DNA topoisomerase and RecF proteins.

並列關鍵字

Transcription ； DNA topoisomerases ； Activation-induced deaminase (AID) ； R-loop ； Gene recombination ； RecF pathway ； RecA ； Nucleoid-associated proteins

參考文獻

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