Mechanistic Insight through Irreversible Inhibition: DNA Polymerase θ Uses a Common Active Site for Polymerase and Lyase Activities
Mechanistic Insight through Irreversible Inhibition: DNA Polymerase θ Uses a Common Active Site for Polymerase and Lyase Activities
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DOI：
10.1021/jacs.8b04158
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发表时间：
2018-07-25
影响因子：
15
通讯作者：
Greenberg, Marc M.
中科院分区：
文献类型：
--
作者：
Laverty, Daniel J.;Mortimer, Ifor P.;Greenberg, Marc M.
DNA聚合酶（Poltheta）是一种多功能酶。它在正常细胞中不是必需的，但它在癌细胞中的上调与细胞对氧化损伤的抵抗和不良预后相关。Pol theta具有聚合酶活性和表征较差的裂解酶活性。我们检查了Pol theta裂解酶在各种脱碱基位点上的活性，并确定在尝试去除通常与C4 '-氧化（pC 4-AP）相关的氧化脱碱基位点时，该酶失活。DNA损伤对Pol theta的共价修饰使得能够确定负责裂合酶反应中希夫碱形成的主要亲核试剂（Lys（2383））。与其他一些碱基切除修复聚合酶不同，Pol theta使用单个活性位点进行聚合酶和裂解酶活性。Lys（2383）突变显著降低两种酶的活性，但不降低DNA结合。Lys（2383）是聚合酶和裂解酶活性所需的证明表明该残基是Pol theta的阿喀琉斯之踵，并提出了设计这种有吸引力的抗癌靶点的抑制剂的前进道路。
DNA polymerase theta (Pol theta) 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 theta possesses polymerase activity and poorly characterized lyase activity. We examined the Pol theta 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 theta by the DNA lesion enabled determination of the primary nucleophile (Lys(2383)) responsible for Schiff base formation in the lyase reaction. Unlike some other base excision repair polymerases, Pol theta uses a single active site for polymerase and lyase activity. Mutation of Lys(2383) significantly reduces both enzyme activities but not DNA binding. Demonstration that Lys(2383) is required for polymerase and lyase activities indicates that this residue is an Achilles heel for Pol theta and suggests a path forward for designing inhibitors of this attractive anticancer target.