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標題: | 探討類核體相關蛋白在R-loop誘發之基因不穩定性及抗藥性機制中所扮演的角色 Investigate the role of nucleoid-associated proteins in the R-loop-mediated genome instability and SOS-mediated drug resistance |
作者: | Cheng-Hsueh Lin 林承學 |
指導教授: | 李財坤 |
共同指導教授: | 鄧述諄,林敬哲 |
關鍵字: | R-loop,SOS response,抗生素抗性,類核體相關蛋白,DNA拓樸異構?, R-loop,SOS response,antimicrobial resistance,Nucleoid-associated proteins,DNA topoisomerases, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 細胞中,被認為含有RNA和DNA互補配對及單股形式存在的非模板DNA結構被稱為是R-loop,此結構可能會引起DNA受損而造成基因不穩定性。因此,細胞演化出許多機制像是RNase H來調控R-loop在細胞中的量。此外,有研究顯示在原核生物中,DNA受損後會緊急啟動「SOS反應」來修復DNA損傷部位來維持基因體的完整性;然而由於錯誤傾向修復聚合酶也會在SOS反應中被誘發,造成修復後的基因突變,進而可能導致細菌對於抗生素產生抗性的原因之一。在我們的研究,我們首先利用S9.6單株抗體以及Sγ3(含好發R-loop的DNA序列)質體造成的死亡現象來探討細菌中R-loop的含量。我們發現:和實驗室之前結果吻合,在topA10突變菌株中,R-loop含量比野生型來的多;而在細菌體內額外表達RNase H時,細菌體內的R-loop含量則會比額外表達突變的RNase H來的少。接著,我們發現R-loop可以造成LexA蛋白的分解以及造成「細胞長絲化(cellular filamentation)」的現象,暗示著R-loop可能為SOS反應上游的來源之一或是R-loop可能會引起DNA損傷;然而,有趣的是,在topA10菌株中,我們卻沒有看到這樣的現象,意指TopA對SOS反應的活化也扮演重要角色。另外,我們在本論文中呈現了第一個證據表示細菌中擁有比較多的R-loop含量會對於trimethoprim以及奎寧類抗生素有比較低的敏感性(即提高抗生素抗性),而相同地,當我們在細菌中額外表現RNase H來減少細胞中的R-loop含量時,就會增加細菌對於trimethoprim以及奎寧類抗生素的敏感性(即降低抗生素抗性)。以上結果闡明兩個假說:R-loop可以活化SOS反應,進而導致抗生素的抗性;另外,TopA和RNase H藉由負向調控R-loop的生成,壓抑SOS反應的產生(但,TopA也對SOS反應的活化扮演重要角色),進而可能調節細菌對抗生素的抗性。最後,由於R-loop的形成和DNA拓樸結構有關,而我們先前實驗室發現不只拓樸異構酶,類核體相關蛋白也會參與在調控DNA拓樸結構中,因次我們也利用了含有氯奎寧的膠體電泳來觀察含不同類和體相關蛋白的菌株菌株中報告質體之DNA超螺旋變化來看類核體相關蛋白,像是HU以及IHF,在R-loop生成中所扮演的角色。綜合我們以上的結果,我們發現R-loop可以作為SOS反應的啟動來源,並第一次提供實驗證據表示R-loop也可能造成SOS反應衍生、引起的抗生素抗性。綜合先前我們實驗室以及本論文結果,我們認為類核體相關蛋白也可能參與在R-loop生成中,這也暗示著這些蛋白也可能在維持基因完整性和R-loop造成的抗生素抗性中扮演一些角色。有趣的是,細胞長絲化的現象和臨床上發現抗抗生素的大腸桿菌之產生及大腸桿菌的輻射抗性有關,因此我們相信此論文的發現對目前未解決的抗生素抗性問題提供了一個解答方向,非常值得進一步研究。 R-loop, which is a cellular structure composing of an RNA/DNA hybrid and a displaced single-stranded DNA with two single and double-stranded junctions, could be a potent source causing genome instability. This genome instability has been suggested to be induced by the ability of R-loop to introduce DNA damage. Hence, cells have evolved various mechanisms to prevent excessive co-transcriptional R-loop formation such as RNase H enzyme to dissolve specifically the RNA/DNA hybrid. Many studies in prokaryotic cells have suggested that DNA damage initiates/activates the “SOS response” for boosting DNA repair capacity in order to maintain genomic integrity; however, with expression of repair polymerase and subsequent elevated level of error-prone replication, SOS response might result in the antibiotic resistance. In our study, correlated with our lab’s previous results, we first found that the cellular R-loop level in the topA10 mutant is higher than that of wild-type strain. In agreement, overexpression of RNase H can effectively suppress the amount of R-loop in cells, which is evidenced by direct detection with the advent of S9.6 antibodies in vitro and the Sγ3 (containing R-loop-prone sequence)-plasmid-mediated lethality in cells. Notably, our results revealed that R-loop can not only introduce the degradation of LexA protein but also be responsible for the phenotype of cellular filamentation, indicating a potential role of R-loop as a resource of SOS response and/or that R-loop formation leads to DNA damage. However, both the topA mutation and overexpression of functional RNase H in cells could restrict this mechanism. These data suggested a complicated regulation of SOS response that in addition to the negative regulatory role of R-loop formation and corresponding activation of SOS response, TopA also plays a direct role in activation of SOS response. Third, our results showed the first evidence that cells with higher cellular levels of R-loop have a reduced sensitivity to trimethoprim and quinolone antimicrobials (i.e. higher antibiotic resistance) and in agreement, reducing the cellular levels of R-loop by plasmid-mediated expression of RNase H in cells can then increase the sensitivity to these antimicrobials (i.e. lower antibiotic resistance). These observations suggested novel notions, those are supported by literature reports, that R-loop can activate SOS response thus leading to the subsequent antibiotic resistance. In addition, possibly through negatively regulation of the R-loop level inside a bacterial cell, TopA and RNase H suppress SOS response and antibiotic resistance. Last, our previous studies also found that in addition to topoisomerase I, nucleoid-associated proteins (NAPs) could also effectively influence the DNA topology. With the postulation that the factors involved in changing the topology and structure of DNA may participate in the regulation of R-loop formation, we further explored the potential role of NAPs such as HU and IHF in R-loop formation by the supercoiling assay. In sum, our results implicated that R-loop plays as a potential role in activating DNA damage-related SOS response and subsequently introducing the SOS response-mediated antimicrobial resistance. Although the TopA deficiency caused an elevated level of R-loop in cells, it is noted that our and other results also suggest TopA is also critically involved in the activation of SOS response. Thus, in the presence of TopA mutant, excess R-loop formation cannot activate SOS response and thus conferring hypersensitivity of topA mutant cells to antibiotics. Furthermore, factors involved in organization of nucleoid DNA also participate in the regulation of R-loop formation, suggesting that they may contribute to maintenance of genome integrity and play a potential role underlying antibiotic resistance. The importance of our findings to the emergent antibiotic resistance needs further investigation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20495 |
DOI: | 10.6342/NTU201702559 |
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顯示於系所單位: | 微生物學科所 |
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