2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/651191摘要：遺傳物質DNA 在生理狀態下會有相當程度的去胺基化，亞黃嘌呤就是由腺嘌呤去胺基而產生，而去胺基的現象可能因為是自發性，也可能是核酸暴露於游離輻射，紫外線，或是亞硝酸而催化，另一方面亞黃嘌呤也可能是來自於核酸複製時誤裝上dITP。若是DNA 上的亞黃嘌呤未被修復，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內切酶五，會將錯誤鹼基３'第二個磷雙酯鍵切開，我們實驗室最近發展出一套修復測定系統，利用限制酶測定dI-dG 及dI-dT 配對錯誤中的dI(亞黃嘌呤)是否被修復，不但可以用來測定細胞萃取液的修復活性，也可以用在純化蛋白系統中進行修復機制的探討。研究結果發現核酸內切酶五系統是大腸菌修復亞黃嘌呤的主要機制。我們也以人類細胞萃取液進行修復測定時，初步結果發現有一個以上的機制可以修復亞黃嘌呤，特別核酸配對修復系統，對於不同環境下的亞黃嘌呤會有選擇性修復的能力，值得做進一步的研究。計畫的第一年，將是進一步研究大腸菌核酸內切酶五參與的修復反應，特別是修復酵素間的蛋白-蛋白交互作用的機制，試著找出所有可能參與反應的酵素。第二年則進行人類細胞萃取液對dI-dG 及dI-dT 的修復研究，並與大腸菌的修復特性進行比較分析。第三年則是針對人類細胞不同修復系統對於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) in DNA can arise fromdeamination of deoxyadenosine, which can be spontaneous or promoted byexposure of DNA to ionizing radiation, UV light, or nitrous acid. Hypoxanthine canalso be introduced by mis-incorporation of dIMP. Hypoxanthine in DNA can pair withcytosine during replication resulting in A:T to G:C transitionsDNA repair is a very important for maintaining genetic integrity in livingorganisms. 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 evolutionaryconserved enzyme that recognizes deaminated bases in DNA. This endonuclease Vincises the DNA at the second phosphodiester bond 3’ to the dI lesion, leaving a 3’OH and a 5' P termini. We recently developed a functional assay with a dI-dG anddI-dT mispair embedded in a restriction endonuclease recognition site. In vitro assaywith cell extracts and re-constitutive assay of purified proteins concluded thatendonuclease V mediated repair is the major pathway to remove the dI lesion in therepair process.However, preliminary results from human cell extracts assay showed morethan one pathways can repair dI lesion efficiently. More interestingly, humanmismatch repair pathway repairs dI lesion only in specific environment, which mayimplicate with the generation of dI lesion.To get better understanding how the dI lesion is repaired in living organism, forthe first year, we plan to perform detail analysis of endonuclease V-mediateddeoxyinosine repair, specifically for the protein-protein interactions of known proteininvolved in the reaction and searching for other components possibly involved in therepair pathway. For the second year, the repair assay of dI-dG and dI-dT with humancell extracts in comparison with repair activity from E. coli extracts. For the third year,we will focus on the interaction of mismatch repair pathway, base excision pathwayand human endonuclease V pathway during the processing of dI-dG and dI-dT lesionand the implication for their selectivity.亞黃嘌呤替代切除修復核酸內切酶五核酸配對修復系統試管中修復分析hypoxanthine, Inosine, alternative excision repairendonuclease VMMR invitro repair