2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/651198摘要：遺傳物質DNA上的驗基，在生理狀態下會有少量的去胺基化，亞黃嗓吟即腺嗓吟去胺基而產生， 在DNA上稱為去氧肌嘌呤核苷（dI)，而核酸暴露於游離輻射，紫外線，或亞硝酸之下也會進一步催化 去胺基。DNA上的亞黃嘌呤若未被修復，在下一次的核酸複製時，就會產生與胞嘧啶配對，進而引發 dA:dT轉dG:dC的點突變。核酸修復對於維護遺傳穩定極為重要，生物體演化出多種系統可以移除DNA的錯誤，亞黃嘌呤被 認為會被亞黃嘌呤核酸糖解酶移除，但是在細菌及哺乳動物中另被找出一種核酸内切酶五，會將錯誤 鹼基3’第二個磷雙酯鍵切開，我們實驗室所發展出修復測定系統，利用限制酶測定dI-dA，dI-dG及 dI-dT配對錯誤中的dI(亞黃嘌呤）的修復，我們分別以細菌萃取液及純化蛋白質重組進行研究，結果 發現核酸内切酶五系統是大腸菌修復亞黃嘌呤的主要機制。在成功的完成細菌系統的分析後，我們試著將此試管中測定延展至人類系統，我們以人類細胞萃 取液進行修復測定初步結果發現，有多個機制參與修復亞黃嘌呤，特別核酸配對修復系統對不同環境 下的亞黃嘌呤會有選擇性修復的能力。核酸修復活性的差異會聯結到個體及族群對疾病的易致病性，許多核酸修復的缺陷與癌症的發 生有關聯。為了能夠更為認識亞黃嘌呤在人類細胞中如何被修復，我們提出二年的研究，在計晝在第 一年，是將我們設計的測定平台，在人類細胞萃取液的測定的最佳化，分別對dI-dA，dI-dG及dI-dT 三種致突變性的損傷進行修復研究，並選取若干已知核酸修復缺陷細胞株進行測定，將其修復特性進 行比較分析。第二年則是針對人類細胞不同修復系統對於dI-A，dI-dG及dI-dT的選擇性修復，研 究不同系統間蛋白-蛋白交互作用，包括已知的核酸配對修復系統對dI-dT的反應，以及其它尚未鑑 定出來的修復活性。<br> Abstract: DNA is subjected to deamination at a physiologically significant rate. Deoxyinosine (hypoxanthine deoxyribonucleoside, dI) in DNA can arise from deamination of deoxyadenosine, which can be spontaneous or promoted by exposure of DNA to ionizing radiation, UV light, or nitrous acid. Hypoxanthine in DNA can pair with cytosine during replication resulting in A:T to G:C transition mutations.DNA repair is a very important for maintaining genetic integrity in living organisms. Specific mechanisms for removal of deaminated bases have evolved. Deoxyinosine in DNA was thought to be removed by hypoxanthine DNA glycosylase, which has been detected in both prokaryotic and eukaryotic organisms. However, several bacterial, archaeal and eukaryotic organisms contain an evolutionary conserved enzyme, endonuclease V (endoV) that recognizes deaminated adenine in DNA. This endonuclease V incises the DNA at the second phosphodiester bond 3’ to the dl lesion, leaving a 3’ OH and a 5' P termini. We developed a functional assay using restriction enzyme scoring to detect repair of A-I, G-I or T-I mispair. The in vitro assay with cell extracts and re-constitutive assay of purified proteins concluded that endonuclease V mediated repair is the major pathway to remove the dI lesion in the repair process.After the successful bacterial study, we also tried to extend the in vitro assay to human system. Our preliminary results from human cell extracts assay showed there may be more than one pathways can process dI lesions. More interestingly, human mismatch repair pathway repairs dI lesion only in specific environment, which may implicate with the generation of dI lesion.Variations in DNA repair activities are connected to both individual and population disease susceptibility. Several DNA repair defects have been linked to cancers. Oxidatively damaged DNA and its repair are also demonstrated positive correlation in colon carcinogenesis.To get better understanding how the dI lesion is repaired in human cells, we propose a 2-year project; for the first year, we plan to extend our established repair assay of A-I, G-I and T-I to human cell extracts in comparison with repair activity from E. coli extracts. For the second year, we will focus on the interaction of human endonuclease V pathway, base excision pathway, and mismatch repair pathway during the processing of A-I, G-I and T-I lesions and the implication for their selectivity.亞黃嘌呤試管中人類核酸修復分析替代切除修復核酸内切酶五核酸配對修復系統鹼基切 除修復系統hypoxanthinedeoxyinosinein vitro human DNA repair assayAlternative excision repairendonuclease VDNA mismatch repairBase excision repair