R-loop的調控及其生物功能之探討:
RecF途徑與DNA拓樸異構酶所扮演的角色

伴隨著轉錄延長作用進行時，新生成的RNA如果繼續和模板DNA互補配對，而讓非模板DNA以單股的形式存在，那麼這個與RNA相關的DNA結構就稱為R-loop。然而在細胞內，R-loop的調控機制及其扮演的生理功能仍然是不清楚的。為了尋找並研究胞內調控R-loop的因子，我們以大腸桿菌作為模式系統，在細菌體內大量表達活化誘導胞嘧啶核苷脫氨酶(activation-induced cytidine deaminase; AID)，以刺激產生突變。不同菌株所展現不同的AID刺激突變倍數(AID-stimulated mutagenesis fold; ASM fold) 用來表示這些細胞內存在著不同程度的R-loop。我們在實驗中發現 (i) RecF活化的菌株(RecF-activated strain, JC7623)擁有極高的ASM fold；同時這個ASM fold可以被額外表達的TopA或RNase H所降低，暗示著R-loop結構確實在此菌的ASM過程中存在。(ii) RecF活化的菌株展現許多獨特的表現性狀，包括質體造成的細胞死亡(plasmid-mediated lethality)、逃跑式DNA複製(run-away DNA replication)以及細胞長絲化(cellular filamentation) ; (iii) 最重要的是，所有的特徵都可以利用送入表達TopA或RNase H來消弭或減少他們展現的程度; (iv) 其中，當我們額外送入兩種活性相反的酵素表達時(TopA及gyrase)，細胞長絲化的現象也完全相反(分別是減少絲化及增加長絲)。綜合以上，我們在本研究中發現RecF蛋白(或RecF途徑)可能有幫助R-loop形成或維持其結構的功能，而過多的R-loop可能就是造成RecF活化菌株展現這些獨特性狀的原因。藉著在分子層級對RecF蛋白、RNase H以及不同DNA拓樸異構酶的操作，我們的研究結果暗示了R-loop的生物功能在DNA複製模式的轉換、DNA損傷訊號、以及DNA重組和細胞存活上的重要性。

關鍵字

R-圈； RecF途徑； DNA拓樸異構酶活化誘導胞嘧啶核苷脫氨酶

並列摘要

The R-loop structure, an RNA-DNA hybrid with a single-stranded DNA region, has been observed during transcription elongation. However, the regulation and biological functions of R-loop still remain largely unknown. In this study, we took advantage of the bacterial genetic model system to solve these problems. We have found that several cellular factors, such as RecF, RNase H and DNA topoisomerases, are involved in the regulation of R-loop formation and thereby modulating its associated cellular functions. Forced expression of human activation-induced deaminase (hAID) in bacteria was utilized to stimulate mutagenesis and the stimulated folds were then used to represent the different levels of R-loop in various strains and conditions. Specifically, the involvement of RecF pathway is supported by the following observations: (i) The fold of AID-stimulated mutagenesis (ASM) in RecF-activated strain JC7623 (recBC sbcBC) was much higher than those measured in other related mutants; furthermore, additional expression of RNase H or TopA suppressed the ASM fold. (ii) Plasmid-mediated lethality, run-away plasmid replication and cellular filamentation phenotype were also exclusively observed in JC7623 cells. (iii) Most importantly, all these phenotypes could be suppressed by ectopic expression of functional RNase H and TopA. (iv) Over-expression of TopA and gyrase reduced and enhanced the filamentation phenotype in RecF-activated bacteria, respectively. In sum, our results suggested that formation of excess R-loops contributes to all the phenotypes observed in RecF-activated JC7623 cells. Furthermore, we have also revealed many novel functions of R-loop in recombination, DNA replication, cellular filamentation and lethality.

並列關鍵字

R-loop ； RecF pathway ； DNA topoisomerase ； Activation-induced deaminase (AID)

參考文獻

Anderson DG & Kowalczykowski SC (1997a) The recombination hot spot chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme. Genes Dev 11, 571-581.

Anderson DG & Kowalczykowski SC (1997b) The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a chi-regulated manner. Cell 90, 77-86.

Arnold DA & Kowalczykowski SC (2000) Facilitated loading of RecA protein is essential to recombination by RecBCD enzyme. J Biol Chem 275, 12261-12265.

Asai T & Kogoma T (1994) D-loops and R-loops: alternative mechanisms for the initiation of chromosome replication in Escherichia coli. J Bacteriol 176, 1807-1812.

Baker TA & Kornberg A (1988) Transcriptional activation of initiation of replication from the E. coli chromosomal origin: an RNA-DNA hybrid near oriC. Cell 55, 113-123.

國際替代計量

R-loop的調控及其生物功能之探討: RecF途徑與DNA拓樸異構酶所扮演的角色

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