Department of Chemistry , Johns Hopkins University , 3400 North Charles Street , Baltimore , Maryland 21218 , United States.
J Am Chem Soc. 2018 Jul 25;140(29):9034-9037. doi: 10.1021/jacs.8b04158. Epub 2018 Jul 12.
DNA polymerase θ (Pol θ) is a multifunctional enzyme. It is nonessential in normal cells, but its upregulation in cancer cells correlates with cellular resistance to oxidative damage and poor prognosis. Pol θ possesses polymerase activity and poorly characterized lyase activity. We examined the Pol θ lyase activity on various abasic sites and determined that the enzyme is inactivated upon attempted removal of the oxidized abasic site commonly associated with C4'-oxidation (pC4-AP). Covalent modification of Pol θ by the DNA lesion enabled determination of the primary nucleophile (Lys) responsible for Schiff base formation in the lyase reaction. Unlike some other base excision repair polymerases, Pol θ uses a single active site for polymerase and lyase activity. Mutation of Lys significantly reduces both enzyme activities but not DNA binding. Demonstration that Lys is required for polymerase and lyase activities indicates that this residue is an Achilles heel for Pol θ and suggests a path forward for designing inhibitors of this attractive anticancer target.
DNA 聚合酶θ（Polθ）是一种多功能酶。它在正常细胞中是非必需的，但在癌细胞中的上调与细胞对氧化损伤的抵抗力和不良预后相关。Polθ 具有聚合酶活性和特征不明显的核酸内切酶活性。我们研究了 Polθ 在各种脱碱基位点上的核酸内切酶活性，并确定该酶在试图去除与 C4'-氧化（pC4-AP）相关的氧化脱碱基位点时失活。DNA 损伤对 Polθ 的共价修饰使我们能够确定在核酸内切酶反应中负责形成席夫碱的亲核试剂（Lys）。与其他一些碱基切除修复聚合酶不同，Polθ 用单个活性位点同时进行聚合酶和核酸内切酶的反应。Lys 的突变显著降低了两种酶的活性，但不影响 DNA 结合。证明 Lys 对于聚合酶和核酸内切酶活性都是必需的，这表明该残基是 Polθ 的阿喀琉斯之踵，并为设计针对这一有吸引力的抗癌靶点的抑制剂提供了一条途径。
