一、分子信號及FRET技術應用於核酸醣解酶之測定評估；二、鎳金屬抑制人類細胞核酸配對錯誤修復機制之分析

Abstract

一 當DNA遭受一些自發性反應或是外源性攻擊，導致特定的DNA 損傷，如產生uracil及hypoxanthine等，就會由鹼基移除修復系統（base excision repair）來進行修復以維持生物體的穩定性。先由特定的核酸醣解酶（DNA glycosylase）辨認及移除受到破壞的鹼基，因此核酸醣解酶是開啟鹼基移除修復系統的關鍵酵素，所以發展出一個快速評估鹼基移除修復系統中核酸醣解酶活性的方法是有其重要性的。 分子信號（molecular beacon）是近年來發展出有用的分析工具，這是一段5’端有fluorophore、3’端有quencher的單股DNA，單獨存在下會形成stem-and-loop的結構，這時給予fluorophore激發光，因為stem結構的關係，fluorophore跟quencher距離接近，使得fluorophore的能量會被quencher吸收，這時分子信號不會發出螢光；如果分子信號stem的結構被破壞，fluorophore在激發後就會發出螢光信號。我們分別設計其中含有尿嘧啶（uracil）以及inosine的分子信號，以測定尿嘧啶-核酸醣解酶（uracil-DNA glycosylase）以及次黃嘌呤-核酸醣解酶（hypoxanthine-DNA glycosylase）的活性。結果發現以純化的酵素進行反應，皆可得到良好的活性與螢光強度線性關係，然而進一步以細胞萃取液進行實驗，則發現有核酸酶（nuclease）活性的干擾。我們另行設計利用螢光共振能量轉移（fluorescence resonance energy transfer；FRET）的原理來設計出一個cFRET探針和cFRET-I5探針，希望藉此探針的設計髮夾結構以避免細胞萃取液中核酸酶的干擾，結果純化的酵素可以得到良好的線性關係，細胞萃取液則依舊有核酸酶干擾結果。 這是一種有用而簡單的測定工具，有待解決的問題是要能找出避免細胞萃取液中核酸酶干擾的方法，以期方便的測定核酸醣解酶的活性。

二 核酸配對錯誤修復系統（Mismatch repair；MMR）廣泛存在於生物之中，對於維持生物基因體的穩定扮演了重要的角色，其中研究最為透徹的是大腸桿菌的核酸配對錯誤修復，參與的蛋白包括了MutS、MutL和MutH，大腸桿菌核酸配對錯誤修復蛋白的同源物也陸續在真核生物的酵母菌與更高等的哺乳動物中被發現，如hMutSα、hMutSβ、hMutLα 。鎳是存在於香菸中的微量金屬，近年來有學者發現鎳是DNA修復反應的抑制劑，長期抽煙的人會容易因為香菸中化合物攻擊DNA而引起突變，進而引起肺癌，而鎳會抑制核苷酸移除修復系統（nucleotide excision repair；NER），所以使得體內的DNA損傷無法修復；過去實驗室已經證實大腸桿菌的核酸配對錯誤修復系統會受到鎳等重金屬的抑制，並且推測出鎳的抑制反應是抑制在修復反應的上游nicking的步驟；另外，也證實了人類的核酸配對錯誤修復系統也會受到鎳的抑制，然而還沒研究證實會抑制在核酸配對錯誤修復反應的哪個步驟。所以為了進一步探討鎳抑制人類核酸配對錯誤修復系統的路徑，我們利用了鹼性瓊脂凝膠分析修復反應的中間產物。結果得知，鎳抑制修復反應的核酸移除（excision）之前的步驟，而且也觀察到鎳會抑制核酸聚合酶的活性，目前的研究結果提供了探討鎳抑制機轉的方向，未來可更進一步的針對專一蛋白或步驟進行分析，藉以更明瞭鎳抑制人類核酸配對錯誤的機制。

1. The base excision repair (BER) pathway plays a key role in protecting the genome from DNA damage. In BER, DNA glycosylases are the critical enzymes that recognize and remove damaged and/or mispaired bases from DNA. Current methods to measure enzyme activities are mostly indirect and time-consuming. Thus, it is very helpful to develop a direct and rapid method to assay DNA glycosylases. Here we introduce a method based on molecular beacon (MB) DNA probe. MBs are hairpin-shaped nucleic acid probes labelled with a 5’-fluorophore and a 3’-quencher in which the fluorophore is held in close proximity to the quencher. Following removal of the modified base of the MB, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Two modified beacons containing of uracil and hypoxanthine have been used to validate the assay on purified proteins with success. However, while applying the assay to cell extracts, the results were interfered by non-specific nuclease activities. Then we design a cFRET probe based on fluorescence resonance energy transfer (FRET) which might be reflectory non-specific nuclease digestion in cell extracts. However, we demonstrated the purified protein successfully and cell extracts still cleaved cFRET probes non-specifically. We hope we can discover an approach to escape interference of nuclease in cell extracts and we can monitor DNA glycosylase activities in real-time. In conclusion, molecular beacons and FRET probes are promising tools for monitoring DNA glycosylase activities in real-time. However, a stable measure need to be developed to avoid the interference of non-specific nuclease activities in cell extracts. 2、 Mismatch repair (MMR) stabilizes the genome by correction of DNA biosynthetic errors, by ensuring the fidelity of genetic recombination, and in mammalian cells by participation in the cellular response to some classes of DNA damage. Escherichia coli mismatch repair has been reconstituted using purified components. Analysis of nuclear extracts of human cells has indicated a similar excision repair mechanism for nick-directed mismatch correction in higher cells, and several reconstituted systems that rely on purified human proteins have been described, such as hMutSα、hMutSβ、hMutLα. Recently, scientists discovered that nickel (II) could be inhibitors of DNA repair. Nickel can greatly enhance the mutagenicity and genotoxicity of compounds in cigarette by inhibiting the nucleotide excision repair pathway in human cells. We have confirmed that nickel could inhibit MMR of E. coli and human and presumed that nickel inhibited repair before MutH incision of the MMR in E. coli. However, there is no evidence of mechanism of nickel inhibiting MMR in human cells. To investigate the inhibitory patch of nickel, we analyzed intermediates of limiting repair reaction by denaturing gel electrophoresis. We found that nickel inhibited repair before excision and nickel could inhibit the activity of DNA ligase. These results provide us a way to demonstrate the inhibitory mechanism. We can focus on the specific protein or the specific step of MMR to clarify the mechanism of nickel inhibiting human MMR.