第五型核酸內切酶及第一型核酸聚合酶校正外切酶處理亞硝酸傷害之生物學意義

Abstract

DNA 的腺嘌呤 (adenosine) 經自發性水解或遭受紫外光、亞硝酸鹽等外源性傷害，會進行脫胺作用變成亞黃嘌呤 (deoxyinosine, dI)。若生物體內的亞黃嘌呤核酸未被修復，經過複製可能造成 A:T→ G:C 突變產生，具高致突變性。目前已知大腸桿菌的亞黃嘌呤核酸主要由第五型核酸內切酶 (Endonuclease V, Endo V) 起始修復。先前本實驗室利用噬菌體 M13mp18 衍生的異雙股核酸，以不同基因突變之大腸桿菌萃取液及純化蛋白系統探討試管中亞黃嘌呤核酸之修復反應，結果顯示僅需 Endo V、第一型去氧核醣核酸聚合酶 (DNA polymerase I, DNA Pol I) 以及連接酶 (DNA ligase) 即可完成修復，我們推論在 Endo V 產生缺口 (nick) 後，由 DNA Pol I 3’端往 5’端核酸外切酶 (3’-5’ exonuclease, 3’exo) 活性負責移除 dI，最後再由 DNA ligase 修補缺口完成修復。 為了印證細菌體中 Endo V 與 DNA Pol I 3’exo對於 dI 修復的必要性，我們以亞硝酸鈉致突變試驗進行測試，缺乏 Endo V 及 DNA Pol I 3’ exo 活性之菌株分別處理亞硝酸鈉，再利用抗生素鏈黴素 (streptomycin) 篩選 rpsL 基因突變產生的菌落數量，評估亞硝酸鈉引發高突變現象在 nfi 突變株及 polAexo突變株與其各別野生株的差異，結果顯示亞硝酸鈉引發的突變率， nfi 突變株是野生株的14倍； polA3’exo 缺失菌株突變的情形是野生株的 57倍，扣除自發性突變後仍為野生株的5倍左右，顯示 DNA Pol I 3’exo 活性及 Endo V 對於處理亞硝酸鈉傷害同樣具重要之生理意義。分別挑取 30 個生長於抗生素培養基的單一菌落，進一步分析亞硝酸鈉處理後 rpsL 基因突變情形，我們發現缺乏 Endo V 的突變菌株，在胺基酸序列43位置發生 A→C 突變比例上升 49%；而 DNA Pol I 中 3’ exo 功能缺失的菌株，序列位置 43 上 A→C 的突變比例增加了 33%，A→G 突變數量多了 43%，說明在亞硝酸鈉的壓力下除了引發 A→G 突變增加，且 Pol I 3’exo 產生 A→C 突變比例和 Endo V 有相同提升的趨勢。 細菌致突變試驗的結果顯示突變菌株和野生菌株的差異趨勢和實驗室先前試管中的結果一致，Endo V 和 DNA Pol I 3’ exo 活性對 dI 之修復角色具有生物學上之意義。

The highly mutagenic lesion, deoxyinosine (dI) can be produced in DNA spontaneously, and the process is enhanced by exposing DNA to ionizing radiation, UV light or nitrous ion. In Escherichia coli, deoxyinosine is excised through repair pathway that is initiated 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-dI, T-dI and A-dI substrate in vitro. In order to evaluate the biological significance of endonuclease V and DNA polymerase I in processing nitrous acid-induced deaminated lesions, we employed nitrous mutagenesis assay to E. coli nfi (endonuclease V deficient) and polAexo (DNA Pol I 3’exonuclease deficient) strains to further understand the importance of these gene products in mutation prevention of nitrous enhanced deamination. Cells were treated with NaNO2 and measured the frequency of streptomycin-resistant mutations in the rpsL gene. The nfi mutant demonstrated 14 times higher mutation frequency than its isogenic wild type. The average frequency of mutants was 57-fold higher in the polAexo strain (KA796 D424A) than that of the wild type strain under nitrous stress. The mutants from the nitrite treatment were collected, and the rpsL gene was sequenced. The most common point mutation after treatment was an A:T→ C:G transvertion. This type of mutation accounted for 97% of total mutation in nfi mutant strains and 60% of total mutation in polAexo mutant strains. The results of mutagenesis assay demonstrated that 3’-5’ exonuclease of Pol I is the integral part of Endo V pathway to protect E. coli against nitrous acid-induced mutagenesis.