單一核苷酸延伸法和質譜儀應用於第一型DNA聚合酶校正活性分析

Abstract

DNA聚合酶校正活性在DNA複製忠誠度上扮演非常重要的角色。傳統偵測DNA聚合酶校正能力的方式包含放射線同位素標定和以電泳為基礎之分析方法。然而，這些方法普遍耗時且過程繁瑣，需要安全措施保護避免暴露於放射線。螢光法也被應用於校正活性的酵素動力分析，像是具有螢光特性的鹼基類似物2-Aminopurine (2-AP)，其可被DNA聚合酶合成至DNA序列與鹼基形成配對，因此被廣泛用於試管中研究聚合酶校正活性。然而，2-AP除了可和thymidine形成配對外，也可能會與cytosine形成熱動力穩定配對，因而降低此方法的特異性，此外，有些DNA聚合酶會選擇性排斥2-AP:T。近年發展出的G-quadruplex也被應用於DNA聚合酶校正活性分析，但其序列設計上有一定的限制性。 MALDI-TOF質譜儀偵測DNA的高解析力已被應用於PinPoint assay，亦即使用沒有任何標記的雙去氧核醣核苷酸三磷酸(ddNTPs)進行引子延伸反應，隨後以MALDI-TOF質譜儀分析，得知基因特定位置的多形性。我們將這個概念延伸應用於聚合酶校正能力之分析。於此，我們設計了一種不需要任何同位素或是螢光標記，且簡單易做的方法測定校正活性。以寡核苷酸引子和模板股黏合，形成引子3’端不同的配對錯誤，DNA聚合酶在有ddNTPs或是一種dNTP的狀態下進行校正，校正過程中切除錯誤配對鹼基的中間產物和重新合成正確鹼基的產物經去鹽類處理後，進入MALDI-TOF質譜儀分析，透過引子質量變化鑑別鹼基配對錯誤是否被校正。 測試Klenow fragment (KF)對引子internal mismatch校正能力實驗中，發現隨著單一鹼基配對錯誤位置改變，校正的效能也有差異，在相同的反應條件下，位於引子3’端2至4個核苷酸位置的鹼基配對錯誤有45%~35%校正效能；然而距離引子3’端5個核苷酸位置的鹼基配對錯誤只剩9%校正效能，伴隨35%單一核苷酸延伸產物，位於引子3’端6至9個核苷酸位置的鹼基配對錯誤沒有明顯的校正產物被觀察到(<1%)。以質譜儀觀察KF對距離引子3’端兩個核苷酸位置之dI分別與A、C、T、G形成的配對錯誤校正情形，發現校正效能為T-I≧G-I >A-I >C-I，推測與鍵結穩定度有關。比較以單一dNTP或ddNTPs作為校正反應材料，發現前者校正效能較佳。以氯化銨(NH4Cl)取代含氯化鈉(NaCl)的試劑進行校正反應可降低頻譜圖雜訊；以鹽酸(HCl)取代酚(Phenol)作為校正反應終止方法，使得整體實驗流程更加簡單快速。在分析過程發現，此種透過質量改變來分析校正效能的方法，不僅可以探討校正效能也可觀察到中間產物，從中透露校正機制訊息，使得這個方法應用性更加廣泛。

Proofreading activity of DNA polymerase is very important factor in maintaining the high fidelity of genetic information during DNA replication. Traditional methods for detection of DNA polymerase proofreading ability include gel-based assays and radioisotopic labeling. However, these methods are generally time-consuming and labor-intensive and require necessary safety measures to avoid radioactive exposing. Fluorometric techniques have been used in kinetic analyses of proofreading activities. Intrinsically fluorescent nucleotide analogs such as 2-aminopurine (2-AP), which are incorporated into the product by DNA polymerase, have been extensively employed to study polymerase proofreading activity in vitro. However, these methods suffer from low specificity due to the ability of thermodynamically stable base pair formation between 2-AP and cytosine. Moreover, some DNA polymerases are able to selectively discriminate the 2-AP:T base pair from the normal A:T base pair. The high resolution of MALDI-TOF MS (matrix-assisted laser desorption ionization time of flight mass spectrometry) for DNA detection has been employed in the PinPoint assay in which the primer extension reactions with four unlabeled ddNTPs followed by MALDI-TOF analysis identify the polymorphism at a given locus. We propose the concept of this approach can be modified to study DNA polymerase proofreading. Herein, we designed a non-labeled and non-radioisotope simple method to measure proofreading. An oligonucleotide primer is annealed to a template DNA forming a mismatched site and is proofread by a DNA polymerase in the presence of all four dideoxyribonucleotide triphosphates or single deoxyribonucleotide triphosphates. The proofreading excision intermediates and re-synthesis products of single base extension are denatured, desalted and subjected to MALDI-TOF mass spectrometry. The proofreading at the mismatched site is identified by the mass change of the primer. We examined proofreading of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus can be proofread efficiently. Internal single mismatches can also be proofread at different efficiencies, with the best correction for mismatches located 2 to 4 nucleotides from the primer terminus. For mismatches located 5 nucleotides from the primer terminus, there was partial correction and extension. No significant proofreading was observed for mismatches located 6 to 9 nucleotides from the primer terminus. Moreover, this method can apply to deoxyinosine (dI) proofreading assay as well. For deamination product of deoxyinosine at penultimate position of primer 3’ ends, all of A-I, C-I, G-I, and T-I can be processed by Pol I proofreading exonuclease, with efficiency being T-I≥G-I>A-I>C-I. The correction efficiency might be determined by the base-pair stability at primer-template junctions. We also tested the reaction using NH4Cl instead of NaCl in reaction buffer. We found Klenow fragment retains the same activity and a much improved MS spectra with less background noise. A protocol using HCl for reaction termination could avoid tedious phenol extraction process and should be amendable for automation for mass screening. In parallel, the mass spectrometric method also has the capability to study both excision intermediates and proofreading products making this assay highly versatile.