Proofreading of insertion/deletion error in slipped strand DNA replication

• Main Authors: Neng-An Chou, 周能安
• Other Authors: Woei-horng Fang
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/jasnar

碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 107 === Insertion/deletion mutation is one of common point mutation during DNA replication . The occurrence of insertion/deletion mutation was thought due to repeated sequence being prone to slippage during DNA replication, so that the replicated product will gain or loss a few nucleotides.i.e. insertion/deletion errors. The insertion/deletion errors in replication can be corrected by the proofreading activity of the polymerase. Previous in vitro DNA synthesis study using template with simple repetitive sequences found that the proofreading efficiency of the polymerase is affected by the loop out position within repeat sequence, being the further the loop out occurs far away from the 3'' end, the less extend to proofread. It was suggested insertion/deletion loops embed upstream of the primer may not be proofread since the matched paring of primer template junction tend to be stabilized and can be successfully extended by DNA polymerase. In order to provide direct evidence for DNA polymerase producing insertion/deletion loop during slipped DNA replication as well as insertion/deletion loop proofreading efficiency of insertion/deletion loop, this study employed proofreading proficient and deficient Klenow polymerase to react with repeat sequences containing oligonucleotides. The resulting products were analyzed by MALDI-TOF MS assay. This study designed DNA substrates with different number (2 to 10) of repeating A．T pairs in primer-template junction. In reactions using proofreading deficient Klenow fragment (3''→5'' exo-) and imbalanced dNTPs pool containing only dATP , this study found that mis-incorporation of A occurred; and the higher number of A．T repeat the greater extend of mis-incorporation in 20 min. Kinetic analysis was performed to confirm above reactions. In comparison, no mis-incorporation were found in the reaction using proofreading proficient klenow as well as reactions using imbalanced dNTPs pool containing dCTP, dTTP, dGTP but not dATP. We then examined proofreading of the Klenow fragment (KF) with DNA containing single nucleotide insertion/deletion loop at different position. Followed by MALDI-TOF MS analysis, the proofreading at the insertion/deletion site is identified by the mass change of the primer. The result indicated that insertion/deletion error within 4 nucleotides upstream from primer terminus could be proofread effectively. We further tested the proofreading efficiency to insertion/deletion loop in slippage strand using DNA substrates containing insertion/deletion loop in nine to fourteen repeating A．T pairs sequence. In reactions using proofreading proficient klenow fragment in the presence of dATP, dCTP and dGTP, we found that the insertion/deletion error substrate with nine to fourteen repeating A．T pairs can be proofread.