| Summary: | 碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 100 === DNA carries genetic information in all organisms. During DNA replication, it is important to maintain genomic fidelity. Three correlating events operate in maintaining the high fidelity of genome：The first is base selection. The second is the proofreading activities of DNA polymerases, which can remove the last mismatched DNA at the primer-template junction. The third is DNA repair systems.
To date, there is no evidence showing that the mismatched DNA at penultimate site of the primer can be edited by DNA polymerase I. Our previous study showed that the proofreading activity of DNA polymerase I could edit deoxyinosine-containing heteroduplex DNA after processing by endonuclease V which created a strand breakage at the second phosphodiester bond 3’ to the deoxyinosine. To figure out how it works, we constructed twelve heteroduplex DNA containing single mismatch at penultimate site of the primer and analysed the proofreading activity.
The involvement of nick translation activity of DNA polymerase I was eliminated. Our results showed that all the twelve heteroduplex DNA can be edited by proofreading activity of DNA polymerase I and there were no general roles for trend of ionic strength in our proofreading assay. We identified purine．purine, the most frequently misinserted mismatches, could be edited well. According to the structure analysis, two large purine bases cause considerable strand strain that may lead to proofreading efficiency elevated. However, purine．pyrimidine mismatches were poorly edited probably due to these structures were similar to the correct Watson-Crick base pairs with minor distortion but the C-A could be edited well.
Furthermore, the mismatch repair system had high efficiency to repair purine．pyrimidine mismatches can compensate to poorly proofreading activity. On the other hand, the large purine bases have increased stacking ability and the common N7 groups may be preferred to bind with the amino acid residue of exonuclease site. We found that the misbase on the primer strand had the more efficiency of proofreading activity but the T-G was not.
Besides, we identified that gap-form substrate had better proofreading activity than nick-form. After removing the wrong base, DNA polymerase I will undergo polymerization. As a result of DNA carrying out polymerization without 5’ to 3’exonuclease activity with the gap-form substrate, it has higher proofreading efficiency.
Conclusively, we identified the proofreading activity of DNA polymerase I can edit DNA mismatches at the penultimate site of the primer. In addition to our previous study, the DNA polymerase I actually could edit deoxyinosine-containing heteroduplex DNA which containing a strand breakage at the second phosphodiester bond 3’ to the deoxyinosine.
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