大腸桿菌第一型DNA聚合酶校對活性活體試驗分析

Abstract

在大腸桿菌中第一型DNA聚合酶為高忠誠性聚合酶，具有校對活性為3’端往5’端核酸外切酶，除了進行DNA複製外，也與許多DNA修復路徑相關，因此其校對活性相當重要。 先前時實驗室建立了MALDI-TOF MS的第一型DNA聚合酶校對活性試驗，結果發現當錯誤配對發生距離3’端1至4個核苷酸位置時可被第一型DNA聚合酶校對，發生距離3’端超過6個核苷酸位置時則無法被校對。至今仍未有報告DNA聚合酶校對活性之活體內試驗，以印證試管中實驗的生物學意義，因此本篇研究致力於建立細菌體內試驗的方法，以與DNA聚合酶體外校對試驗比對。 本篇研究以pBluescript II SK(+) phagemid載體製備在5’端帶有C:C標誌，以及3’有錯誤配對之G:T或C:C錯配含三個核苷酸之gap受質及C:C錯配之nick受質，將受質轉型至菌株NM522(polA WT)進行校對活性的評估，同時也以大腸桿菌KA796(polA+)及無3'→5'核酸外切酶活性菌株KA796D424A (polAexo D424A mutation)比對校對活性。 首先，以3’端第一個核苷酸為G:T錯配之三個核苷酸gap受質測定第一型DNA聚合酶錯誤配對校對能力，再由限制酶水解判讀校對結果，發現KA796(55.33%)與KA796D424A(49.33%)校對活性無顯著差異，懷疑為mismatch repair system(MMR)所干擾，因此將G:T錯配換成C:C錯配，以降低MMR影響。接著，以NM522、KA796及KA796D424A測定3’端第一個核苷酸為C:C錯誤配對之gap受質皆有高的背景值，C:C錯配結構會影響其周圍配對之穩定性造成strand loss增加，而校對活性結果NM522(51.33%)、KA796(52.67%)、KA796D424A (44%)無顯著差異，可能為第二型與第三型DNA聚合酶之干擾，因此設計nick受質以降低除了第一型DNA聚合酶以外的聚合酶干擾。以NM522測定3’端第一個核苷酸位置C:C錯配之nick受質，C:C錯配之nick受質(74%)較C:C錯配之gap受質(51.33%)顯示出較高的校對活性且有統計上的差異(p=0.008)以及較低的背景值，但仍因C:C結構於3’末端位置不利於DNA連接酶作用，造成引子股損失增加，卻只觀察到少數的無校對修復結果。而3’端第四個核苷酸位置C:C錯誤配對之nick受質於NM522結果發現無校對成功結果，其可能受到DNA連接酶之干擾，因此後續需將受質之5’端磷酸根去除，以避免受質在未受到DNA聚合酶反應前就被黏合。實驗也觀察到aberrant結果，可能為受質進行轉型作用引起SOS反應使DNA polymerase I*增加，因其在複製過程中較容易插入錯誤核苷酸造成。 本研究顯示於NM552結果中當錯誤配對位於3’端1、2、3個核苷酸位置時可被第一型DNA聚合酶校對，但校對比率呈下降趨勢(1:74%、2:64%、3:56.6%)。KA796與KA796D424A之結果比對發現，無3'→5'核酸外切酶活性菌株KA796D424A其校對比率下降，但仍有校對成功結果懷疑為proofreading in trans影響，為KA796D424A內其他具有核酸外切酶活性蛋白與第一型DNA聚合酶進行校對反應。因此，C:C錯配且去除5’端磷酸根之受質可使用於野生型菌株NM522進行後續研究生物體內第一型DNA聚合酶校對錯誤配對與否於3’端距離之臨界點與試管試驗結果是否相符，而於KA796與KA796D424A之校對活性比對則因仍無法排除proofreading in trans之干擾，需後續實驗進行改善。

DNA polymerase I is a high-fidelity polymerase in Escherichia coli with 3’→5’proofreading activity. DNA Pol I plays important roles in DNA replication and various DNA repair pathways, and it is crucail in maintain gene stability. We had established a MALDI-TOF MS based proofreading activity analysis for DNA polymerase I in vitro. The results revealed that mismatch within the primer-template junction about 1 to 4-nt from the primer 3’end provoked proofreading, mismatch at 6-nt away from the primer 3’terminus escaped from proofreading. To date, there have been no reports of in vivo proofreading assay for DNA polymerase to validate the biological significance for in vitro data. Thus, this study aims to establish a phagemid-base substrates for in vivo Pol I proofreading assay, moreover, to compared the in vitro proofreading assay results. In this study, synthetic oligonucleotide-cloned pBluescript II SK(+) phagemid containing C:C marker at 5’end substrates, with a 3-nucleotide gap containing G:T or C:C mismatch, or a nick containing C:C mismatch. For evaluating proofreading activity of Pol I, mismatched substrates were transformed into E.coli wild-type strain NM522(polA WT). The assay also performed in PolI proofreading proficient strain KA796(polA+) and PolI proofreading deficient strain KA796D424A(polAexo D424A mutation). First, the proofreading activity of DNA Pol I was measured by using 3-nucleotide gap containing G:T substrate. The proofreading activity showed no significant difference in strain KA796(55.33%) and KA796D424A(49.33%), suggesting that mismatch repair system (MMR) may interfere the proofreading assay. To minimize MMR interference, the G:T mismatch was replaced by a C:C mismatch. However, the C:C mismatch gapped substrates caused high strand loss background in strain NM522, KA796, and KA796D424A, possibly due to the structure destabilization by the C:C mismatch surrounding base pairing. Moreover, no significant difference in proofreading activity among NM522 (51.33%), KA796 (52.67%), and KA796D424A (44%), possibly due to interference by DNA polymerase II and III. We then designed a nicked substrates that would reduce interference by Pol II and Pol III. The proofreading activity in NM522 with the gapped C:C substrate (51.33%) and the nicked C:C substrate (74%) showed significant difference (p=0.008). Moreover, the quick ligation by DNA ligase to the nicked substrates with C:C mismatch at the fourth nucleotide from 3’ end showed no proofreading results. Therefore, removing the 5' phosphate group from the substrate is solution to prevent premature ligation before proofreading reaction This study demonstrates that proofreading activies of C:C errors from different positions at primer-template junction showed different efficiencies in the NM522, being highest 74% at 3'-end, 64% at penultimate site, and 56.7% at 3rd position from the primer 3' end and the proofreading rate decreases in KA796D424A. Therefore, the C:C mismatch nicked substrates lacking the 5' phosphate group is suitable for in vivo studies in strain NM522 to investigate the proofreading patch of the 3' end, which shown a comparable results to the in vitro experiments.