- 學位論文
建構含次黃嘌呤之噬菌粒受質以進行試管中及體內第五型核酸內切酶修復分析
Construction of phagemid-based deoxyinosine substrate for both in vitro and in vivo endonuclease V repair assay
摘要
細胞中的腺嘌呤(Adenine; A)會因發生自發性的脫胺作用或受到外源性的氧化壓力等傷害而變成次黃嘌呤(Hypoxanthine; Hx)。以Hx為鹼基與五碳醣結合，則稱為次黃嘌呤核苷酸(Deoxyinosine；dI)。第五型核酸內切酶修復系統(Endonuclease V repair pathway)是大腸桿菌中主要修復次黃嘌呤核苷酸的修復途徑之一。本實驗室先前已做過試管內次黃嘌呤核苷酸修復實驗，得知只需要第五型核酸內切酶、第一型DNA聚合酶、DNA連接酶即可完成整個修復反應。 我們希望能以細胞體內修復試驗印證此路徑，但本實驗室先前分別以噬菌體(M13mp18)以及質體(pUC18)作為載體所設計的T-I 核酸受質在進行細菌體內試驗時效果皆不理想。 因此本研究選擇使用噬菌粒(pBluescript II SK)作為載體，製備含T-I mismatch的核酸受質，期望此全新設計的核酸受質可同時適用於試管中以及細菌體內修復試驗，將此核酸受質分別轉型到BW25113 (WT)、JW5547 (nfi-) 、KA796 (polA+)、KA796 D424A (polA exo-)，四株不同的大腸桿菌中，進行細菌體內修復試驗。結果可看出JW5547 (nfi-)之修復效率較其野生株大腸桿菌低，而KA796 D424A (polA exo-)之修復效率和其野生株大腸桿菌相比並無差別，這和先前以質體為載體做出的偽陽性修復現象是相符的，我們懷疑是第五型核酸內切酶造成DNA缺口後，第一型DNA聚合酶大次單元無法移除錯誤片段完成修復，進而使DNA複製叉崩壞所造成的現象，因此我們無法判定核酸受質有無被修復成功，我們稱為偽陽性修復現象，因此本實驗室同時使用以噬菌粒作為載體之核酸受質，多設計一個C-C錯誤配對之標記，以區別偽陽性修復與陽性修復。 此外，本研究亦使用以噬菌粒作為載體之T-I核酸受質進行試管中純化蛋白質系統試驗，結果顯示在缺乏第一型核酸聚合酶中的3’-5’核酸外切酶活性的組別，其修復效率相較其他組別有明顯下降，證明了本實驗室對於第五型核酸內切酶修復系統路徑的推論，主要由第一型核酸聚合酶中3’-5’核酸外切酶的活性負責移除含有dI片段核酸。
並列摘要
The highly mutagenic deoxyinosine (dI) lesion can be produced in DNA spontaneously, and is enhanced by nitrous ion exposure. In Escherichia coli, dI is repaired through endonuclease V (EndoV) repair pathway. Our previous in vitro assay demonstrated that EndoV, DNA polymerase I (Pol I), and E. coli DNA ligase was sufficient to reconstitute the dI repair. To find out gene products requirement for Endo V repair pathway, we employed both bacteriophage-based (M13mp18) dI-containing substrate and plasmid-based (pUC18) dI-containing substrate for in vivo repair assay. However, the results of both approaches were unsatisfactory. In this study, we developed a phagemid-based new substrate containing a T-I mismatch that can be used in both in vivo assay and in vitro assay. The results of T-I mismatch substrate demonstrated the similar repair level as in previous plasmid-based dI-containing substrate study: the repair level of nfi mutant was much lower than its isogenic wild type. However, the pol I proofreading exonuclease deficiency strain KA796 D424A (polA exo-) showed as high repair level as in its isogenic KA796 (polA+). It’s suspected a persistent single strand break was generated due to incomplete repair in KA796 D424A (polA exo-); and in subsequent plasmid replication the dI containing strand was lost because of replication fork collapse. Therefore, only continuous template strand survived the replication and was scored by our assay. This hypothesis was confirmed by using substrate containing both T-I and strand discrimination marker C-C mismatch to determine true repair level. We also used the same substrate for in vitro assay with purified proteins. The result demonstrated that the repair efficiency of the test without polymerase I 3’-5’ exonuclease activity decreased dramatically compared to its isogenic wild type. This observation confirmed our hypothesis and provided solid evidence to support Pol I proofreading exonuclease is the major enzyme activity to remove dI lesion in Endo V repair pathway.