R-loop形成之拓樸調控機制研究

Abstract

當細胞進行轉錄時，去氧核醣核酸（DNA）的拓樸結構會隨之改變：當核糖核酸聚合酶（RNA polymerase）向前移動進行轉錄時，前方的DNA構型將累積正向超螺旋（positive supercoils），而在轉錄機制後方的DNA則會形成負向超螺旋結構（negative supercoils）。新生成的RNA偶有機會與模板DNA形成配對，此時這個結構包含RNA-DNA雜交體（hybrid）、以及裸露出的非模板單股DNA，稱為R-loop。根據我們實驗室與其他科學家先前的研究，即使R-loop在細胞的DNA複製、轉錄、及基因表達上扮有相當重要的角色，過多R-loop的存在卻也可能造成基因不穩定性而危害細胞。因此，細胞中有許多調控R-loop生合成的因子。本論文使用大腸桿菌作為模式生物系統，去探討具解決DNA負向超螺旋功能的DNA拓樸異構酶I（topoisomerase I, TopA）以及具解決DNA正向超螺旋功能的DNA旋轉酶（gyrase），對於R-loop調控的作用機轉。為此目的，我們使用了以下四種方法去檢測R-loop的形成：（i）使用純化的S9.6抗體來直接地偵測細胞內RNA-DNA雜交體的多寡；（ii）在細菌中大量表現活化誘導胞密啶核苷脫氨酶（activation-induced cytidine deaminase, AID），利用其作用於R-loop上單股DNA將胞嘧啶（cytosine）轉變為尿嘧啶（uracil）的特性，求得AID誘發突變倍數（AID-induced mutagenesis fold, ASM fold）；（iii）細菌絲狀生長現象（bacterial filamentation）；以及（iv）質體DNA超螺旋檢測（supercoiling assay of plasmid reporter）。如同我們所預期，我們發現在TopA缺陷的細胞有較高的S9.6訊號及較高的ASM fold，顯示TopA可能藉由其解決負向超螺旋之功能，在R-loop的調控上扮有抑制之角色。此外，在累積過多的染色體損壞而未修復的情況下，細菌會啟動SOS response，表現大量參與染色體修復的基因，延遲細胞分裂，使細菌免於死亡，此反應會造成細胞的型態改變，產生絲狀生長的現象。與R-loop可能導致DNA損壞的概念一致，我們發現在TopA缺陷的細胞中確實有絲狀生長的現象。此外，由於負向超螺旋可能導致R-loop，而R-loop的生成將有機會伴隨著過度負向超螺旋（hyper-negative supercoil）。我們亦發現在TopA缺陷的狀況下會產生過度負向超螺旋的現象。綜合以上結果，我們認為TopA在抑制R-loop的形成扮有重要的角色。接著，在一隻gyrase溫度敏感的菌株RFM445 [gyrB203 (Ts) gyrB221 (CouR)]中，我們發現相較於在攝氏28度時的表現型，其在37度時的表現型（較低的gyrase活性）有較高的RNA-DNA雜交體訊號、細菌絲狀生長、以及過度負向超螺旋現象。總結來說，我們的結果顯示過多的R-loop可能導致細胞絲狀生長、較高的突變機率、以及過度負向超螺旋現象。TopA作用於抑制R-loop生成，並在維持基因穩定性（genome instability）扮演重要的角色，而gyrase對於R-loop的影響仍需進一步的研究。

During transcription, the topological conformation of double-helical DNA changes: As RNA polymerase moving forward, the DNA segment ahead of RNA polymerase is constrained with positive supercoils, and the DNA part behind transcription machinery becomes negatively supercoiled (i.e. defined as the “twin-domain supercoiling model”). Occasionally, the nascent RNA inserts into negatively supercoiled DNA helix and thus hybridizing to the template DNA. This structure is called R-loop, containing the RNA-DNA hybrid, non-template ssDNA, and junctions of ssDNA and dsDNA regions. Based on our and other previous studies, though R-loop plays critical roles in DNA replication, transcription and gene expression, but it is also a hazardous source with excess amount of R-loop that may lead to genome instability and poor cell growth. Therefore, there are factors in cells tightly regulate the R-loop formation. Here, using bacteria E. coli as a genetic model system and according to twin-domain supercoiling model, we decided to focus first on the topological regulatory enzymes such as DNA topoisomerase I (TopA) and gyrase, which can resolve negative and positive supercoils respectively. To detect the R-loop formation, we had employed the following 4 assays: (i) S9.6 antibody; (ii) AID-stimulated mutagenesis (ASM); (iii) bacterial filamentation and (iv) reporter DNA supercoiling assays. Specifically, the S9.6 antibody recognizing the RNA-DNA hybrid was used as a direct tool to detect R-loops. The ASM assay was established by ectopic expression of AID to induce deamination of cytosine to uracil on the single-stranded DNA (ssDNA) portion of R-loop, thus leading to higher mutation rates and ASM fold, those indirectly represent the amount of R-loop. As expected, we found that the TopA-deficient cells have a higher level of S9.6 signal and an elevated ASM fold, suggesting that TopA had a role in suppressing R-loop formation, possibly through its activity in specifically resolving negative supercoils. It is known that DNA damage activates SOS response, increases expression of repair factors and arrest of cell division (causing a filamentation phenotype). Consistent with the notion that R-loop might lead to DNA damage and activation of SOS response, we had found that TopA deficiency resulted in cellular filamentous morphology. Also, since negative supercoiling might allow the formation of R-loop, it is presumable that R-loop formation is accompanied with hyper-negative supercoils. We found that TopA deficiency appeared to be hyper-negatively supercoiled in supercoiling assay. In sum, we suggested that TopA plays a dominant role in the suppressing of R-loop formation. Surprisingly, in a gyrase temperature-sensitive mutant strain RFM445 [gyrB203 (Ts) gyrB221 (CouR)], the elevated RNA-DNA hybrid signal, cellular filamentation phenotypes and hyper-negative supercoiling were found in a higher cultivation temperature (37°C, presumably a lower gyrase activity condition) compared to the lower one (28°C). However, the cellular filamentous phenotype might also result from the gyrase deficiency and corresponding DNA replication, chromosome segregation problems … and so on. In conclusion, our results thus suggest that excess amount of R-loop might lead to elevated cellular filamentation, higher mutagenesis rate and hyper-negative supercoiling phenotypes. TopA functions mainly in inhibition of R-loop formation and thus plays a significant role in maintaining genome stability, while the functional role of gyrase on R-loop still needs further investigations.