DNA聚合酶I於引子不同位置配對錯誤鹼基校正活性分析

Abstract

DNA為生物體之遺傳物質，其高度複製忠誠度（fidelity）有助於維持基因穩定性及預防突變發生。DNA聚合酶主要透過三種方式降低複製錯誤率：鹼基選擇性配對（base selection）、3端往5端外切酶（3’→5’ exonuclease）之校正能力（proofreading activity）及錯誤配對修復機制（mismatch repair system）。研究指出DNA聚合酶能夠移除引子末端鹼基或末端連續兩個錯誤鹼基。本實驗室發表文獻 (DNA Repair 9: 1073-1079)指出當DNA聚合酶I與DNA內切酶V（endonuclease V）、DNA連接酶（DNA ligase）及dNTP共同存在下能夠修復G-dI的錯誤配對，應是由DNA內切酶V切斷dI上游第二個磷酸雙酯鍵，活化DNA聚合酶I校正活性導致。本實驗室進一步分析DNA聚合酶I對斷股上游第二個鹼基為錯誤配對十二種受質之校正活性。發現DNA聚合酶I可以修復斷股上游第二個錯誤配對（林千如, 國立臺灣大學101學年碩士論文），顯示斷股上游第二個位置有錯誤配對會活化DNA聚合酶I的3端往5端外切酶活性。然而我們對DNA聚合酶I校正外切酶作用於引子3’端上游配對錯誤的整體修復能力仍未全然明瞭，為了探究DNA聚合酶I的3端往5端外切酶的活化能力，我們設計一系列含配對錯誤的受質，各受質與引子3’端相隔1至8個正常配對鹼基以檢測聚合酶往3’上游配對錯誤的校正活性。配對錯誤受質的的設計，是利用限制酶對於序列具專一性，來偵測所製備的配對錯誤是否被校正。我們將配對錯誤鹼基設計在限制酶作用的位置，然後安排不同的限制酶在配對錯誤鹼基的下游，以利產生3’端不同位置的斷股。如此便製備出在斷股上游不同位置有配對錯誤鹼基的核酸受質。我們設計A-A 及T-T兩種具有代表性的配對錯誤進行測試，將各種不同位置配對錯誤受質與DNA聚合酶I作用，並在含有0.1 mM dNTPs 的情形下觀察其校正活性。此外，為觀察酵素反應作用初期的反應狀況，我們將不同的核酸受質與DNA聚合酶I反應，將反應時間設定在6分鐘之內，觀察酵素動力學的變化，並比較不同位置的配對錯誤，核酸修復效率與反應速率的差異。實驗結果發現，DNA聚合酶I能夠校正至斷股上游第四個錯誤配對之異雙股DNA，在不同濃度的DNA聚合酶I具有不同的校正活性。我們也發現對於不同位置錯誤配對的核酸受質，DNA聚合酶I的校正活性與反應速率也有所差異。利用線性配對錯誤受質與DNA聚合酶I作用的校正活性，排除DNA聚合酶I行缺口轉譯（nick translation）的可能性後，我們確認DNA聚合酶I是利用3’端往5’端外切酶，對斷股上游錯誤配對鹼基進行校正。此結果與先前X光晶體繞射研究核酸在DNA聚合酶活化位之轉移的推論相符合，這些結果對於DNA聚合酶在校正機制（proofreading）上的研究，有正面的幫助。另外，本研究也證實，配對錯誤在離斷股3’端上游超過 5個核苷長度時，就不容易被DNA 聚合酶I所移除，這個結果可以作為site-directed mutagenesis 實驗設計寡核苷酸引子(oligonucleotide primer )時的參考。

DNA carries genetic information in all living organisms. During DNA replication, it is important to maintain genomic integrity. Three mechanisms are involved in maintaining the high fidelity of genome. The first is base selection during replication; the second is the proofreading activities of DNA polymerases, which can remove the mis-incorporated nucleotide at the primer-template junction. The third is DNA mismatch repair systems. According to previous studies, it is known that terminal mismatch and consecutive two mismatches at the 3’ end of the primer can be edited by DNA polymerase I (pol I). Our previous study showed that the proofreading activity of pol I could edit deoxyinosine-containing heteroduplex DNA following the process of endonuclease V which create a strand breakage at the second phosphodiester bond 3’ to the deoxyinosine (DNA Repair 9: 1073-9). To figure out how it works, we constructed twelve heteroduplex DNAs containing single mismatch at the penultimate site of the primer and analyzed the proofreading activity. The results showed that all of the twelve heteroduplex DNAs can be edited by proofreading activity of pol I. However, the overall capacity of pol I proofreading exonuclease toward mismatches embedded upstream of the primer is still not fully understood. Therefore, we designed a series of mismatch substrates containing a strand break at 0 to 7 nucleotides 3’ to the mismatch, which mimic mismatches embedded in primer template junctions, to study proofreading activity of pol I. Mismatches were designed to interrupt a restriction endonuclease recognition sequence so that proofreading activity can be scored by the restriction endonuclease assay. We also placed several restriction endonucleases sequences at 3’ side to the mismatches so that in the same sequence content a series of substrates containing different strand breaks can be prepared. The two mismatches, A-A and T-T, were employed for the proofreading assay. The assay condition was in the presence of 0.1 mM each of the four dNTPs to mimic in vivo replication condition. Kinetic reactions of different substrates were assayed in a 6-min reaction span to obtain the initial rates for the comparison of substrate specificity for pol I proofreading. Our results showed that pol I can actively edit mismatches at -1, -2, -3, and -4 positions of the primer terminus. The correction levels were pol I concentration dependent, and also demonstrated certain degree of substrate specificity. Linearized heteroduplex substrate could also be efficiently proofread by pol I which ruled out the possible interference by non-specific nick translation. The results of this study is consistent with previous X-ray crystallography study that at least 4 nucleotide from 3’ end of the primer were required for transfer from polymerization site to exonuclease active site for editing. In addition, we also found mismatches located more than 5 nucleotides from 3’ end were very difficult to remove by proofreading proficient DNA polymerase. The observation could provide a good guidance for designing oligonucleotides for gapped duplex site-directed mutagenesis.