Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5' exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3'-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain-especially the O helix-to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3' exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO-POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

中文翻译：

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分枝杆菌 DNA 聚合酶 I：作为脱辅基酶的 POL 结构域以及与 DNA 引物模板复合物和全长 FEN/EXO-POL 酶的活性和晶体结构。


分枝杆菌 Pol1 是一种双功能酶，由 N 端 DNA 瓣核酸内切酶/5' 核酸外切酶结构域 (FEN/EXO) 和 C 端 DNA 聚合酶结构域 (POL) 组成。在这里，我们记录了 Pol1 的其他功能：RNA:DNA 杂交体的活瓣 RNA 链上的 FEN 活性以及 DNA 引发的 RNA 模板上的逆转录酶活性。我们报道了 POL 结构域的晶体结构，作为脱辅基酶和与 3'-双脱氧末端 DNA 引物模板和 dNTP 的三元复合物。 POL 的拇指、手掌和手指子域与引物模板和传入 dNTP 的三磷酸盐形成广泛的界面。从脱辅基酶的开放构象到三元复合物的接近闭合构象的进展需要拇指和手指模块的几个片段的无序到有序的转变以及手指子域（尤其是O螺旋）的向内运动以接合引物模板和 dNTP 三磷酸盐。分枝杆菌 Pol1 POL 的独特结构特征包括残留 3' 核酸外切酶子结构域中的锰结合位点和蛋白质-DNA 界面处的非催化水桥镁复合物。我们报道了双功能 FEN/EXO-POL 脱辅基酶的晶体结构，该结构揭示了 FEN/EXO 结构域中两个活性位点金属的位置。