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標題: | 核酸內切酶第五型主導之修復系統於生物體內亞黃嘌呤核酸修復之分析 The Repair of Endonuclease V-Mediated Alternative Excision Repair of Deoxyinosine in vivo |
作者: | Pei-Rong Wu 吳佩蓉 |
指導教授: | 方偉宏(Woei-Horng Fang) |
關鍵字: | 亞黃嘌呤核酸,脫胺作用,核酸修復,核酸內切?第五型,第一型去氧核醣核酸聚合?, Hypoxanthine,Deoxyinosine,Endonuclease V,DNA polymerase I, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | DNA中的腺嘌呤經由自發性水解或者是遭受紫外光、亞硝酸等外源性傷害,會進行脫胺作用變成亞黃嘌呤。倘若生物體內的亞黃嘌呤核酸未被修復,核酸複製時傾向將其與dC配對,則可能造成A:T→G:C的突變產生。
目前已知在大腸桿菌中主要是由核酸內切酶第五型(endonuclease V)主導之修復系統負責修復亞黃嘌呤核酸,先前本實驗室利用噬菌體M13mp18衍生的異雙股核酸探討於試管中亞黃嘌呤核酸之修復反應,利用不同基因突變之大腸桿菌的蛋白質萃取液以及純化蛋白的系統來探討亞黃嘌呤核酸所需的因子及其修復機制,結果顯示核酸內切酶第五型、第一型去氧核醣核酸聚合酶(DNA polymerase I)以及去氧核醣核酸連接酶(DNA ligase)參與亞黃嘌呤核酸之修復。首先由核酸內切酶第五型辨識亞黃嘌呤核酸的位置,於其3’端之第二個磷酸雙酯鍵水解產生缺口起始修復反應,研究結果我們推測由第一型去氧核醣核酸聚合酶中3’往5’核酸外切酶活性負責移除亞黃嘌呤核酸,最後由DNA連接酶修補缺口。 為了印證在細菌體中核酸內切酶第五型與第一型去氧核醣核酸聚合酶中3’往5’核酸外切酶活性對於亞黃嘌呤核酸修復的必要性,我們嘗試研究於in vivo中,核酸內切酶第五型與第一型去氧核醣核酸聚合酶中3’往5’核酸外切酶活性對於亞黃嘌呤核酸修復也是不可或缺。我們利用轉形作用將含亞黃嘌呤核酸的異雙股核酸送入含不同基因突變之大腸桿菌勝任細胞,挑出溶菌斑以限制酶鑑定並計數,分別比較野生型與核酸內切酶第五型突變株(nfi)、第一型去氧核醣核酸聚合酶突變株(polA1)以及配對錯誤修復蛋白突變株(mut S)之差異。結果顯示於野生型菌株中可移除亞黃嘌呤核酸的比例約56%,在mut S突變菌株中可移除亞黃嘌呤核酸的比例約58%至62%,由此結果可知配對錯誤修復系統對於修復亞黃嘌呤核酸無關;在缺乏nfi之菌株中只有15%至18%,與野生型菌株相比約差四倍,可知nfi 基因產物對於修復亞黃嘌呤核酸的重要性;而在缺乏polA1之菌株中只有6%至9%,與野生型菌株相比約差八倍,更顯得pol I基因產物在亞黃嘌呤核酸修復路徑中扮演極為重要之角色。 我們in vivo實驗結果顯示突變型菌株和野生型菌株的差異趨勢與先前實驗室in vitro的結果一致,核酸內切酶第五型以及第一型去氧核醣核酸聚合酶在修復亞黃嘌呤核酸中扮演十分重要的角色。 Deamination of adenine can occur spontaneously under physiological conditions, generating highly mutagenic lesion, hypoxanthine (deoxyinosine in DNA). The process is enhanced by exposure of DNA in ionizing radiation, UV light and nitrous ion. If not repaired, hypoxanthine tends to generate A:T to G:C transition mutations. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway by endonuclease V. According to our previous studies, the purified protein system containing Endonuclease V, DNA Polymerase I, and E. coli DNA ligase was sufficient to reconstitute the repair of G-I, T-I and A-I substrate in vitro. To comfirm in physiological condition, endonuclease V and DNA polymerase I are important to deoxyinosine correction in vivo. We therefore constructed deoxyinosine-containing bacteriophage M13mp18 DNA to evaluate its repair in vivo. We designed G-I and T-I in the recognition sequences of specific restriction endonucleases. After transfection of dI-containing phage DNA into E. coli, the correction can be evaluated by restriction digestion of replication form DNA from resulting plaques. E. coli mutS (MutS deficient), nfi (endonuclease V deficient) and polA (pol I deficient) as well as wild type strains were used for repair evaluation. The repair level in wild type E. coli was 55%, and about 60% in mutS mutant. It suggested that mismatch repair plays little role in processing deoxyinosine in vivo. In contrast, the repair level in nfi mutant decreased to less than 18%, and the repair level in polA mutant was further decreased to 8%. These results highly suggested the gene products of nfi and polA are very important for the deoxyinosine repair. The in vivo results are consistent with our previous in vitro study that DNA polymerase I and endonuclease V are the enzymes required for removing deoxyinosine in endonuclease V-initiated repair pathway. |
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