重複序列核酸複製之插入/缺失錯誤校正活性分析

Abstract

插入/缺失突變(insertion/deletion mutation)是DNA複製時常見的點突變之一，發生的原因是因為重複序列DNA在複製過程中容易出現滑動，使得複製產物在重複序列的地方會比模板股增加幾個核苷酸或減少幾個核苷酸，這種情形就是插入/缺失錯誤，在複製過程中插入/缺失錯誤的出現可以經由DNA聚合酶的3'端至5'端外切酶活性進行校正，在先前文獻指出，當DNA發生插入/缺失突變時，DNA聚合酶的校正效能會因為loop out形成位置的不同，而受到影響，也就是loop out出現的位置距離引子股3'端比較遠時，DNA聚合酶會比較傾向於繼續延伸引子股而非進行校正。另外引子股的末端有正確的雙股配對時，會使末端結構比較穩定，可能因此被聚合酶視為正確的受質，而繼續延伸。為了要瞭解DNA聚合酶在插入/缺失突變的生成及校正所扮演的角色，本研究利用MALDI-TOF質譜儀平台來分析Klenow fragment (KF)進行核酸複製時可能產生的插入/缺失錯誤以及校正能力。 首先探DNA討聚合酶在重複A．T序列上產生複製錯誤的情形，以校正能力缺失的Klenow fragment (3'→5' exo-)及分別以末端帶有兩個重複A．T序列及十個重複A．T序列的DNA受質在只加入dATP情形下進行反應，結果發現在末端帶有兩個重複A．T的組別，僅出現少量延伸訊號，但末端帶有十個重複A．T的組別則引子股全部都生成延伸產物，之後分別對末端帶有不同數目A的引子股進行聚合反應，結果發現當重複A．T的數目越多，延伸訊號的比例也會越高，由此結果能知道重複A．T序列的長度確實會影響插入/缺失錯誤的產生，最後再以帶有四、六、八、十個重複A．T序列的組別進行動力學實驗，結果發現有較長重複序列的組別在各個時間點都有較高的延伸訊號，而且在帶有十個重複序列的組別一開始的反應初速度就明顯高過其他組別。 再來是探討DNA聚合酶對插入/缺失錯誤的校正反應，實驗是設計在不同位置上帶有固定位置單一核苷酸插入/缺失錯誤，和Klenow fragment及dATP、dTTP、dGTP及ddCTP進行反應，反應後以鹽酸終止反應，再用質譜儀分析結果以了解校正效能。從實驗結果中發現插入/缺失發生在引子股末端倒數四個以內時，都能有效校正，當插入/缺失發生在引子股末端倒數第五個核苷酸時，則會出現一部分延伸訊號及一部分校正訊號，但如果錯誤發生在倒數第五個核苷酸以外時，則只有延伸訊號出現。 最後本研究也對DNA聚合酶對於發生在重複序列上插入/缺失錯誤的校正情形進行探討，分別設計帶有9~14個重複A．T序列的DNA 受質，也就是slippage strand，且上面帶有缺失錯誤，加入Klenow fragment及dATP、dCTP及dGTP進行反應。結果卻發現在帶有9~14個重複序列上的插入/缺失錯誤都能被校正。

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.