2014-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/651189摘要：遺傳物質 DNA 上的鹼基，在生理狀態下會有少量的去胺基化，亞黃嘌呤就是由腺嘌呤去胺基而產生，在 DNA 上稱為去氧肌嘌呤(dI)，而去胺基的現象也會因核酸暴露於游離輻射，紫外線，或是亞硝酸而進一步催化。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，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。根據本實驗室現有的成果，我們計畫在第一年，將是進一步研究大腸菌核酸內切酶五修復系統中，核酸複製酶 I 所進行修復區段的範圍，這個研究可釐清是否仍有其它內切酸酶參與反應的可能;我們也準備建立一套細胞內實驗及致突變實驗，以確認從我們先前試管中實驗鑑定出的修復蛋白生物學上的意義，這對於建立此修復系統完整模型至為重要。計畫第第二年則將我們的測定平台延伸到人類細胞萃取液的測定，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-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 fromdeamination of deoxyadenosine, which can be spontaneous or promoted by exposureof DNA to ionizing radiation, UV light, or nitrous acid. Hypoxanthine in DNA can pairwith cytosine during replication resulting in A:T to G:C transitions.DNA repair is a very important for maintaining genetic integrity in living organisms.Specific mechanisms for removal of deaminated bases have evolved. Deoxyinosine inDNA was thought to be removed by hypoxanthine DNA glycosylase, which has beendetected 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. Thisendonuclease Vincises the DNA at the second phosphodiester bond 3’ to the dI lesion, leaving a 3’OH and a 5' P termini. We developed a functional assay usingrestriction enzyme scoring to detect repair of dI-dA, dI-dG or dI-dT mispair. The in vitroassay with 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. Our 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 in-depth analysis of the repair patch generated E.coli repair pathway. Specifically focus on the activity demonstrated by DNApolymerase I, which plays dual role of both error excision and gap filling. This studycould further establish that Pol I is the enzyme responsible for excision and ruling outthe possibility that other endonuclease involvement in the repair. We also plan todevelop an in vivo assay and a mutagenesis assay to evaluate the biologicalsignificance for the repair proteins identified by our previous in vitro assay. And it isvery important for building the model of complete repair scheme. For the second year,we plan to extend the repair assay of dI-dA, dI-dG and dI-dT to human cell extracts incomparison with repair activity from E. coli extracts. For the third year, we will focus onthe interaction of human endonuclease V pathway, base excision pathway, andmismatch repair pathway during the processing of dI-dA, dI-dG and dI-dT lesion andthe 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hypoxanthineInosinealternative excision repairendonuclease VMMR invitro repair