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2016年8月4日 訊 /生物谷BIOON/ --我們細胞中的DNA會被多種外部因子持續損傷，比如包含菸草煙霧的致癌物或來源於太陽光的紫外線輻射等；如果未被修復，這些損傷就會引發突變，最終就會導致細胞癌變；那麼細胞為何不快速有效地進行DNA損傷的修復呢？為了完成該目的，細胞會利用一系列酶類，而且這些酶類必須同時採取行動才能夠鑑別並且修復基因組的損傷，然而長期以來科學家們往往難以理解上述過程的複雜性及其相關的機制。
感謝納米技術的幫忙，2012年，一組科學家通過深入研究後，實時觀測到了這些酶類修復DNA損傷的過程，隨後研究者們對DNA損傷修復機制的第一步開始深入研究分析，如今研究者再次取得成功，他們首次從整體上揭示了DNA損傷修復的完整過程。
研究者利用了一種特殊類型的顯微鏡，其可以幫助觀察單一分子的DNA和蛋白質，並對其進行操作，這就可以幫助研究者清楚觀察到被紫外線損傷的單一DNA分子的表現情況；隨後研究人員對RNA聚合酶進行分析，該酶負責「閱讀」DNA代碼的長度並且開啟DNA代碼向蛋白質產生的過程，但如果閱讀了損傷DNA的片段就會出現停頓，當然我們應該非常感謝這種所謂的停頓作用，其可以幫助細胞識別DNA的損傷並開啟修復過程，實際上研究者還能夠成功觀察到同RNA聚合酶相互作用的四種蛋白：Mfd，UvrA，UvrB 和UvrC，同時還能夠看到被紫外線損傷的DNA的後期修復過程。
通過確定這些組分發揮作用的順序，以及其在分子接力賽中彼此切換的特性方式，研究者就能夠確定DNA損傷修復過程的關鍵步驟；本文研究也將具有新的應用價值，比如幫助開發抵禦癌症的新療法以及治療病原菌感染等；的確，當癌細胞對化療或者放療產生耐藥性時（這些療法的目的就是破壞癌細胞的DNA），因為癌細胞有著活性狀態的DNA修復機制，以及其經歷著臨床治療產生的DNA損傷。
因此研究者就可以在癌症治療期間通過抑制癌細胞的DNA修復，從而就可以抑制腫瘤對療法的耐受性；本文研究結果表明，某些病原菌，比如引發肺結核的結核分枝桿菌，其會利用和Mfd蛋白非常相似的特殊蛋白來進行增殖，缺乏Mfd蛋白的細菌DNA鏈對於紫外線光導致的損傷具有相當的抗性；因此鑑別出這些蛋白幫助細胞進行DNA修復的工作機制或許對於開發抵禦病原菌感染的新療法將非常重要。（生物谷Bioon.com）
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Reconstruction of bacterial transcription-coupled repair at single-molecule resolution
Jun Fan, Mathieu Leroux-Coyau, Nigel J. Savery & Terence R. Strick
Escherichia coli Mfd translocase enables transcription-coupled repair by displacing RNA polymerase (RNAP) stalled on a DNA lesion and then coordinating assembly of the UvrAB(C) components at the damage site1, 2, 3, 4. Recent studies have shown that after binding to and dislodging stalled RNAP, Mfd remains on the DNA in the form of a stable, slowly translocating complex with evicted RNAP attached5, 6. Here we find, using a series of single-molecule assays, that recruitment of UvrA and UvrAB to Mfd–RNAP arrests the translocating complex and causes its dissolution. Correlative single-molecule nanomanipulation and fluorescence measurements show that dissolution of the complex leads to loss of both RNAP and Mfd. Subsequent DNA incision by UvrC is faster than when only UvrAB(C) are available, in part because UvrAB binds 20–200 times more strongly to Mfd–RNAP than to DNA damage. These observations provide a quantitative framework for comparing complementary DNA repair pathways in vivo.
Initiation of transcription-coupled repair characterized at single-molecule resolution
Kévin Howan, Abigail J. Smith, Lars F. Westblade, Nicolas Joly, Wilfried Grange, Sylvain Zorman, Seth A. Darst, Nigel J. Savery & Terence R. Strick
Transcription-coupled DNA repair uses components of the transcription machinery to identify DNA lesions and initiate their repair. These repair pathways are complex, so their mechanistic features remain poorly understood. Bacterial transcription-coupled repair is initiated when RNA polymerase stalled at a DNA lesion is removed by Mfd, an ATP-dependent DNA translocase1, 2, 3. Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors. These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.
A dynamic DNA-repair complex observed by correlative single-molecule nanomanipulation and fluorescence
Evan T Graves, Camille Duboc, Jun Fan, François Stransky, Mathieu Leroux-Coyau & Terence R Strick
We characterize in real time the composition and catalytic state of the initial Escherichia coli transcription-coupled repair (TCR) machinery by using correlative single-molecule methods. TCR initiates when RNA polymerase (RNAP) stalled by a lesion is displaced by the Mfd DNA translocase, thus giving repair components access to the damage. We previously used DNA nanomanipulation to obtain a nanomechanical readout of protein-DNA interactions during TCR initiation. Here we correlate this signal with simultaneous single-molecule fluorescence imaging of labeled components (RNAP, Mfd or RNA) to monitor the composition and localization of the complex. Displacement of stalled RNAP by Mfd results in loss of nascent RNA but not of RNAP, which remains associated with Mfd as a long-lived complex on the DNA. This complex translocates at ~4 bp/s along the DNA, in a manner determined by the orientation of the stalled RNAP on the DNA.