Construction of phagemid-based deoxyinosine substrate for both in vitro and in vivo endonuclease V repair assay
| Autor: | Wei-Chen Chang, 張煒晨 |
|---|---|
| Rok vydání: | 2017 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 105 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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