X家族DNA聚合酶非典型反應機制之結構基礎

Abstract

長久以來，科學家們認為DNA聚合酶遵循典型反應途徑，即酵素先與DNA結合，再藉DNA模板以Watson-Crick配對準則選擇正確的核苷酸以進行催化合成。然而，近期研究指出部分DNA聚合酶能以非典型反應途徑調節其準確度，即在沒有DNA存在下預先結合核苷酸。人類DNA聚合酶Mu(Pol Mu)是X家族中的特殊成員，具偏好以錳離子(Mn2+)進行催化反應且極低準確度之罕見特性。我們發現Pol Mu在與DNA結合前，不僅以具活性的構型預先結合錳·去氧核苷酸(MndNTP)，且於各種反應初期即顯現其偏好錳離子的特性，例如：在單一缺口DNA或非同源性末端接合(Non-homologous end joining)中將dNTP/rNTP嵌入、將dNTP嵌入單股DNA、將8-oxo-dGTP嵌入相對應之模板dA (錯誤配對)或dC (正確配對)。藉解析19種不同催化態之酵素構型，我們不僅證實了非典型反應機制與Pol Mu的多種功能在相關，也印證了Pol Mu可能以非典型反應途徑作為其主要之催化途徑。另一方面，Pol Beta是X家族中高準確度的DNA聚合酶，其主要功能為鹼基切除修復(Base excision repair)。以酵素結構角度而言，Pol Beta與DNA結合後能有效利用N-helix閉合做為構型選擇機制以配對正確核苷酸。然而，我們發現在沒DNA存在下，Pol Beta對MndNTP有意想不到之親和力。Pol Beta:MndNTP的結構顯示其為70% N-helix閉合狀態且負責與DNA結合的D-subdomain亦有構型之變化。我們認為此具催化活性結構的Pol Beta:MndNTP一但與DNA結合後將促使錯誤配對的發生。此外，這項結果也可為錳離子為何能降低DNA聚合酶準確度之問題提供一種可能的解釋。總體而言，本研究擴展了DNA聚合酶可能的反應途徑，亦即含括典型及非典型兩種反應機制。

DNA polymerases have long been considered to follow the canonical pathway in which enzyme binds DNA first, then chooses the correct nucleotide by Watson-Crick base pairing. However, recent studies have reported that the noncanonical pathway involving nucleotide prebinding in the absence of DNA can modulate the fidelity of some DNA polymerases. The X-family DNA polymerase Mu (Pol Mu) has Mn2+ specificity for its functions and extremely low fidelity in the presence of Mn2+. Here, we found that Pol Mu can prebind MndNTP in a functionally relevant conformation. Besides, the Mn2+ preference has already exerted at the prebinding stage. A total of 19 structures have been determined in this study to evaluate the structural basis for the noncanonical mechanism in various functions of Pol Mu. Our findings suggest that Pol Mu can be preactivated by prebinding nucleotide in the presence of Mn2+ ion. Moreover, Pol Mu could utilize this noncanonical mechanism as the primary pathway for catalysis. On the other hand, Pol Beta, a high-fidelity DNA polymerase in X-family, is the main enzyme for base excision repair. Pol Beta effectively utilizes the N-helix closure as a conformational selection mechanism to choose the correct incoming nucleotide after DNA binding. Our study showed that Pol Beta has an unexpected affinity to MndNTP in the absence of DNA. The structures of Pol Beta in complex with MndNTP exhibit the conformational changes in D-subdomain for DNA binding and a 70% closed state. The catalytically active conformation may promote mismatch pairing once MndNTP-bound Pol Beta binds to DNA. Besides, our finding can also provide a possible explanation for why Mn2+ reducing the fidelity of DNA polymerases. Overall, this work expands the scale of the noncanonical pathway to include DNA polymerases from high to low fidelity.