質譜儀應用於插入/缺失錯誤校正活性分析

Proofreading assay of insertion/deletion loops using MALDI-TOF mass spectrometry

林貴卿(Kuei-Ching Lin)

指導教授 ： 方偉宏

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201802586

摘要

小量核苷酸的插入/缺失是DNA複製時常見的點突變，會引發框架位移的移碼突變且與癌症的發生有關。插入/缺失的產生，常常因為在DNA重複序列的區域容易出現DNA滑動。一般DNA複製時出現錯誤，會由聚合酶進行校正，但是DNA聚合酶對插入/缺失的校正效能，卻會因為重複序列的不同而有所差異。當出現插入/缺失環狀結構，若環的位置距離引子3’端較遠，可能無法被聚合酶的校正活性辨識及修復，而且因為引子股的3’端有正確的雙股配對，末端結構較穩定，可能會被DNA聚合酶視為正確的受質，而繼續做引子延伸。
為了要瞭解校正活性修復插入/缺失環的情形，我們在DNA上設計插入/缺失錯誤進行校正活性反應。在引子股3’末端倒數不同的位置上設計單一核苷酸插入/缺失錯誤，再以Klenow fragment及ddNTP進行校正反應，再以質譜儀分析校正效能。結果發現引子3’末端倒數5個核苷酸以內的插入/缺失錯誤都可以被校正，引子3’末端倒數第6以後位置的插入/缺失錯誤則難以被校正。
接著分別在寡核苷酸序列的模板股以及引子股設計4個、10個與15個重複A-T序列的DNA 受質，也就是滑動股DNA，黏合之後重複核苷酸上會形成單一插入/缺失錯誤環結構，再以Klenow fragment及ddNTP進行校正。發現4個核苷酸重複序列上，單一插入/缺失錯誤可以被Klenow fragment校正。在10個核苷酸重複序列上，單一插入/缺失錯誤同樣傾向形成在靠近引子3’端的地方，大部分可以被校正，若在引子3’末端加上2至3個正確配對的核苷酸使引子末端的結構較穩定，則有少量的插入/缺失錯誤可以逃離校正。在15個重複序列的引子3’端加上3個正確配對的核苷酸，重複核苷酸上的單一插入/缺失錯誤可以形成在遠離引子3’端的內部，可逃離Klenow fragment的校正。
除了校正活性，也使用3 '往5 '端外切酶活性缺乏的Klenow fragment (3'→5' exo-)與dATP、dGTP 、dTTP進行聚合反應，由於Klenow fragment (3'→5'exo-)在缺乏dCTP的情形下聚合酶延伸到模板股的G之前便會停止聚合。結果發現當DNA末端為錯誤配對時，Klenow fragment (3'→5'exo-)無法做引子延伸，表示聚合酶可以偵測引子末端錯誤。當使用10個重複核苷酸上單一插入/缺失錯誤的DNA作為反應受質時，可以觀察到引子延伸訊號，表示在10個重複序列上插入/缺失錯誤可能形成在遠離引子3’端的內部，並且引子末端有多個正確配對的核苷酸使結構穩定，因此聚合酶無法偵測到引子內部的插入/缺失錯誤。

關鍵字

插入/缺失錯誤 ； DNA滑動 ； 聚合酶校正活性反應 ； 聚合酶聚合活性反應 ； Klenow fragment ； 質譜儀

並列摘要

Insertion/deletion mismatch is a common point mutation during DNA replication, being associated to human disease such as cancer. Strand slippage known as repetitive sequence may lead to insertion/deletion mutation. DNA polymerase proofreads errors and the proofreading efficiency depends on the contents of repetitive sequence during DNA replication. It’s been known that insertion/deletion loops occurring upstream from the primer terminus may not be proofread by DNA polymerase since the matched paring of primer terminus will be recognized as correct substrate for extension by DNA polymerase. In order to understand the proofreading efficiency of insertion/deletion loop, we designed oligonucleotides containing insertion/deletion to perform proofreading assay.
We examined proofreading of the Klenow fragment (KF) with DNA containing single nucleotide insertion/deletion loop at different position. Proofreading products were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). The result indicated that insertion/deletion error within five nucleotides upstream from primer terminus could be proofread. The insertion/deletion error from six to nine nucleotides upstream of primer terminus escaped proofread.
We further tested the proofreading efficiency to insertion/deletion loop in slippage strand using DNA substrates containing insertion/deletion loop in four, ten and fifteen repetitive sequence. Adding KF and ddNTPs, we found that insertion/deletion loop in both the four and ten repetitive sequences could be proofread by KF. However addition of two or three matched nucleotides at primer terminus of ten repetitive sequence, some insertion/deletion loops escaped proofreading.
We also examined interference of insertion/deletion mismatch to polymerase activity. We used the KF(3'→5' exo-), dATP, dGTP and dTTP for the assay. In the reaction lack of dCTP, the primer extension of KF (3'→5' exo-) would be terminated at the position opposite to a guanine in template. We found KF(3'→5' exo-) could not extend primer with a terminal mismatch. However, in the ten repetitive sequence containing an insertion/deletion mismatch could be extended by KF(3'→5' exo-). We conclude that when insertion/deletion loop form at the position over four nucleotides upstream from the primer terminus in repetitive sequence could escape proofreading.

並列關鍵字

insertion/deletion loop ； strand slippage ； proofreading activity ； polymerization activity ； Klenow fragment ； MALDI-TOF MS

參考文獻

Bebenek, K., and Kunkel, T.A. (1995). Analyzing fidelity of DNA polymerases. Methods in enzymology 262, 217-232.

Bonk, T., and Humeny, A. (2001). MALDI-TOF-MS analysis of protein and DNA. The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry 7, 6-12.

Calos, M.P., and Miller, J.H. (1981). Genetic and sequence analysis of frameshift mutations induced by ICR-191. J Mol Biol 153, 39-64.

Carver, T.E., Jr., Hochstrasser, R.A., and Millar, D.P. (1994). Proofreading DNA: recognition of aberrant DNA termini by the Klenow fragment of DNA polymerase I. Proc Natl Acad Sci U S A 91, 10670-10674.

Cowart, M., Gibson, K.J., Allen, D.J., and Benkovic, S.J. (1989). DNA substrate structural requirements for the exonuclease and polymerase activities of prokaryotic and phage DNA polymerases. Biochemistry 28, 1975-1983.