大腸桿菌內切酶五系統對亞黃嘌呤修復之試管中研究

Abstract

在一般的生理狀態下，腺嘌呤的脫氨作用會自發性地進行且將其變成亞黃嘌呤。當 DNA 暴露在離子化輻射、紫外光、亞硝酸或是加熱的環境下，會促進此脫氨作 用的進行並且會產生具有高致突變性的脫氧肌苷。倘若 DNA 中的脫氧肌苷未被修 復，則可能會有 A:T→G:C 同類置換的突變產生。在大腸桿菌中，脫氧肌苷的清除 主要是由內切酶五(endonuclease V, endo V)所啟始的修復作用來負責。在本研 究中，我們利用噬菌體 M13mp18 及 f1PM 衍生的異雙股 DNA 來進一步探討脫氧肌苷 的修復反應，而且這些 M13mp18 及 f1PM 的異雙股 DNA 於特定的限制酶辨認序列或 是切位均帶有脫氧肌苷。未被修復的脫氧肌苷異雙股 DNA 可以抵抗其相對應的限 制酶的作用，如此即可利用此特性來評估脫氧肌苷的修復程度。我們利用不同基 因突變的大腸桿菌的蛋白質萃取物及純化蛋白的系統來探討修復脫氧肌苷所需的因子及機制，比較三種帶有脫氧肌苷的異雙股 DNA 受質(A-I, T-I, 及 G-I)的修復 差異。實驗結果顯示 DNA 聚合酶一(DNA polymerase I, Pol I)的 3’端往 5’端 的外切酶活性對於修復脫氧肌苷是相當重要的。為了瞭解 DNA 聚合酶一對於移除 損壞核苷酸的特性，我們系統性地分析 DNA 聚合酶一對於 12 種位於斷股倒數第二個位置的鹼基錯誤配對的校正活性，如此錯誤配對的結構即相當於內切酶五作用 後的中間產物。實驗結果顯示，位於斷股倒數第二個位置的錯誤配對及脫氧肌苷都能夠被修復，這樣的實驗結果為我們的論點提供強烈的支持，即在內切酶五對 受質 DNA 產生斷股後，DNA 聚合酶一的 3’端往 5’端的外切酶活性是主要用來移除位於斷股倒數第二個位置的脫氧肌苷的外切酶。本研究所提出的由內切酶五所主導的脫氧肌苷切除修復的模擬路徑如下所述：首先由內切酶五將脫氧肌苷靠近 3’ 端的第二個磷酸雙酯鍵打斷並啟始修復反應的進行，接著由 DNA 聚合酶一的 3’端往 5’端的外切酶活性將脫氧肌苷等核苷酸移除，核苷酸的缺口再由 DNA 聚合酶一進行聚合，最後則由 DNA 連接酶(ligase)完成修復反應。

Deamination of adenine can occur spontaneously under physiological conditions, and give hypoxanthine. This process is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion deoxyinosine in DNA. Such DNA lesion tends to generate A:T to G:C transition mutation if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, correction of dI lesions was investigated using M13mp18 or f1PM derived heteroduplex containing a deoxyinosine resided in the recognition site or cleavage site of specific restriction endonuclease. Unrepaired G-I heteroduplex was first found to be refractory to the cleavage of corresponding restriction endonuclease markers, therefore G-I was used to evaluate the repair dI lesions. Cell-free extracts from various E. coli mutants were used to investigate the deoxyinosine repair requirement and mechanism. We further compared the repair of three mutagenic deoxyinosine lesions A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3'-5' exonuclease activity of DNA polymerase I (Pol I) was very important for processing the deoxyinosine lesions. To understand the nature of Pol I in removing damaged nucleotides, we systemically analyzed its proofreading activity to 12 possible mismatches 3'-penultimate to a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3' penultimate site were corrected with similar efficiency, which strongly support the idea that the 3'-5' exonuclease activity of E. coli Pol I is the primary exonuclease activity for removing 3'-penultimate deoxyinosines derived from endo V nicking reaction. In our proposed model of endo V-mediated excision repair, the repair of dI is initiated by endo V generated strand break at the second phosphodiester bond 3’ to the dI lesion. The subsequent removal of the damaged base is performed by the 3’-5’ exonuclease activity of DNA polymerase I. Finally, the gap is filled and sealed by DNA polymerase I and DNA ligase respectively.