Baños-Mateos Soledad, van Roon Anne-Marie M, Lang Ulla F, Maslen Sarah L, Skehel J Mark, Lamers Meindert H
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK.
Nat Commun. 2017 Oct 11;8(1):855. doi: 10.1038/s41467-017-00886-w.
High-fidelity DNA replication depends on a proofreading 3'-5' exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues. Cryo-EM analysis reveals the entry path of the primer strand in the PHP-exonuclease active site. Furthermore, the PHP-exonuclease shows a striking similarity to E. coli endonuclease IV, which provides clues regarding the mechanism of action. Altogether, this work provides important insights into the PHP-exonuclease and reveals unique properties that make it an attractive target for novel anti-mycobacterial drugs.The polymerase and histidinol phosphatase (PHP) domain in the DNA polymerase DnaE1 is essential for mycobacterial high-fidelity DNA replication. Here, the authors determine the DnaE1 crystal structure, which reveals the PHP-exonuclease mechanism that can be exploited for antibiotic development.
高保真DNA复制依赖于一种与复制性DNA聚合酶相关的校对3'-5'核酸外切酶。主要病原体结核分枝杆菌（Mtb）的复制性DNA聚合酶DnaE1利用其内在的PHP核酸外切酶，该酶不同于在大肠杆菌和真核复制体中发现的典型DEDD核酸外切酶。PHP核酸外切酶的机制尚不清楚。在此，我们展示了Mtb DnaE1聚合酶的晶体结构。PHP核酸外切酶具有一个三核锌中心，由九个保守残基配位。冷冻电镜分析揭示了引物链在PHP核酸外切酶活性位点的进入路径。此外，PHP核酸外切酶与大肠杆菌内切核酸酶IV有显著相似性，这为作用机制提供了线索。总之，这项工作为PHP核酸外切酶提供了重要见解，并揭示了使其成为新型抗分枝杆菌药物有吸引力靶点的独特特性。DNA聚合酶DnaE1中的聚合酶和组氨醇磷酸酶（PHP）结构域对分枝杆菌的高保真DNA复制至关重要。在此，作者确定了DnaE1晶体结构，揭示了可用于抗生素开发的PHP核酸外切酶机制。
