探討類核體相關蛋白調控R-loop 之機制與其生物意義

Abstract

中文摘要 R-loop是一種雙股去氧核糖核酸(DNA)被新生成的核糖核酸侵入所形成的結構，偶爾會在轉錄延伸的過程中發生。這種結構參與在許多生物的作用之中，例如ColE1質體的複製起始(initiation of ColE1 plasmid DNA replication)、免疫球蛋白體細胞超突變和類型轉換重組(immunoglobulin somatic hypermutation and class switch recombination)及表觀基因調控(epigenetic regulation)等。然而，前人的研究發現R-loop的形成會導致複製叉(replication fork)停滯、轉錄障礙以及基因組不穩定，因此，細胞中具有許多因子會調控R-loop的形成，使其達到動態平衡。不過對於R-loop調控的機制卻仍舊有許多未知的地方。我們利用活化誘導胞嘧啶核苷脫氨酶(Activation-induced cytidine deaminase, AID)來偵測大腸桿菌模式系統中R-loop的形成。AID可作用在R-loop中的單股DNA，將上面的胞嘧啶(cytosine)脫氨化轉變成尿嘧啶(uracil)，因而產生致突變性(mutagenesis)。因此從AID刺激突變機率所計算而得到的倍數(AID-stimulated mutagenesis fold, ASM fold)可用來表示胞內R-loop的多寡。透過這個實驗模式系統，進一步證明DNA拓樸異構酶(DNA topoisomerases)會在核糖核酸聚合酶(RNA polymerase)的前後端維持DNA超螺旋(DNA supercoiling)的結構，我們發現細菌第一型拓樸酶TopA可負向調控R-loop的形成，而第二型拓樸酶gyrase可正向調控R-loop的形成，這也。基於以上發現，我們假設R-loop可以被一群有能力改變DNA拓樸結構的因子所調控，例如DNA拓樸異構酶或是與類核體結合之相關結構蛋白等。在本篇論文中，我們想發掘在大腸桿菌中主要的類核體相關蛋白(nucleoid-associated proteins, NAPs)，其中包含Fis、Hfq、H-NS、StpA、IHF及HU蛋白，在R-loop的調控中所扮演的角色。首先，我們確定在不同NAPs突變株中，質體DNA的拓樸結構的確會受到影響。接下來我們發現IHF可逆向調控R-loop形成，其基因去除可造成較野生型高的ASM倍數、較多的Sγ3表達質體所造成的死亡(Sγ3 plasmid-mediated lethality)以及細菌之細胞延長(cellular elongation)。相反地，HU可正向調控R-loop形成，其基因去除可造成較低的ASM fold、較少的Sγ3質體所造成的死亡以及沒有細胞延長。因此，我們首先發現參與在組成類核體結構中的因子同時可以調控R-loop的形成。我們的研究提供了第一次實驗證據這些類核體相關蛋白因子不只在類核體結構上扮演角色，更可以進一步影響DNA複製起始、轉錄相關重組(transcription-associated recombination)以及基因不穩定性等生物作用。 關鍵詞：類核體；類核體相關蛋白；轉錄；R-loop；活化誘導胞嘧啶核苷脫氨酶；DNA拓樸異構酶；基因不穩定性

ABSTRACT R-loop is a structure of double-stranded DNA invaded by nascent RNA, which forms during transcription elongation occasionally. It has been suggested to play roles in the various biological processes such as the initiation of ColE1 plasmid DNA replication, immunoglobulin class switch recombination and epigenetic regulation. In addition, previous studies have also suggested that excess R-loop could result in replication fork stall, transcription blockage and genome instability. Thus, cells have developed many cellular factors to regulate R-loop formation in a low-level homeostasis. Nevertheless, the underlying mechanisms of the regulation of R-loop remain largely unsolved. We have utilized the mutagenic activity of AID to detect R-loop formation in an E. coli model system. AID deaminates cytosine converting it into uracil on the single-stranded DNA in R-loop and the rendering mutagenesis. Therefore, the mutation fold calculated from AID-stimulated mutagenesis assay (ASM fold) is suggestive of the intracellular level of R-loop. Through this system, we have provided experimental supports for the previous “twin-supercoiling domain” hypothesis that topoisomerases maintain the steady state of DNA supercoiling ahead of and behind transcription machinery and identified type I topoisomerase TopA as a suppressive factor for R-loop formation and type II topoisomerase gyrase as a promoting factor for R-loop formation. Based on above discoveries, we have hypothesized that factors involved in changing the topology and structure of DNA may participate in the regulation of R-loop formation, such as DNA topoisomerase and the nucleoid-associated proteins (NAPs). In this study, we further explored the potential roles of major nucleoid-associated proteins in E. coli, including Fis, Hfq, H-NS, StpA, IHF and HU, in regulating R-loop formation, possibly through altering and maintaining the topology and organization of nucleoid DNA. We have confirmed that the topological state of a reporter plasmid was affected differentially in NAP-deleted strains, suggesting their ability to alter the DNA topology and possibly structure. Using the experimental systems for indicating R-loop formation, we have then identified IHF as a potential negative regulator of R-loop formation, whose gene deletion resulted in higher ASM fold, more Sγ3 plasmid-mediated lethality and bacterial cells to elongation (cellular filamentation). In contrast, HU serves as a potential positive regulator of R-loop formation, whose gene deletion led to lower ASM fold, reduced Sγ3 plasmid-mediated lethality and no cellular filamentation. Hence, for the first time, our results revealed that factors involved in organization of nucleoid DNA, participate in regulation of R-loop formation. Subsequently, through regulating R-loop formation, these factors might then contribute to initiation of DNA replication, transcription-associated recombination, and genome instability. Key words: Nucleoid; Nucleoid-associated proteins; Transcription; R-loop; Activation-induced cytidine deaminase (AID); DNA topoisomerases; Genome instability