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首页> 外文期刊>Biochemistry >The Steric Gate Amino Acid Tyrosine 112 Is Required for Efficient Mismatched-Primer Extension by Human DNA Polymerase κ
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The Steric Gate Amino Acid Tyrosine 112 Is Required for Efficient Mismatched-Primer Extension by Human DNA Polymerase κ

机译：通过人类DNA聚合酶κ有效地错配引物延伸需要Steric Gate氨基酸酪氨酸112

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摘要

Human DNA is continuously damaged by exogenous and endogenous genotoxic insults. Toncounteract DNA damage and ensure the completion of DNA replication, cells possess specialized DNAnpolymerases (Pols) that bypass a variety of DNA lesions. Human DNA polymerase κ (hPolκ) is a member ofnthe Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolκ is characterized by itsnability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N2n-deoxyguanine (BPDE-N2n-dG)nand thymine glycol] and efficiently extend primers with mismatches at the termini. hPolκ is structurallyndistinct from E. coli DinB in that it possesses an ∼100-amino acid extension at the N-terminus. Here, wenreport that tyrosine 112 (Y112), the steric gate amino acid of hPolκ, which distinguishes dNTPs from rNTPsnby sensing the 20n-hydroxy group of incoming nucleotides, plays a crucial role in extension reactions withnmismatched primer termini. When Y112 was replaced with alanine, the amino acid change severely reducednthe catalytic constant, i.e., kcat, of the extending mismatched primers and lowered the efficiency, i.e., kcat/Km,nof this process by ∼400-fold compared with that of the wild-type enzyme. In contrast, the amino acidnreplacement did not reduce the insertion efficiency of dCMP opposite BPDE-N2n-dG in template DNA, norndid it affect the ability of hPolκ to bind strongly to template-primer DNA with BPDE-N2n-dG/dCMP. Wenconclude that the steric gate of hPolκ is a major fidelity factor that regulates extension reactions fromnmismatched primer termini.
机译：人类DNA受到外源性和内源性遗传毒性损害的不断破坏。为了对抗DNA损伤并确保DNA复制的完成，细胞具有专门的DNAn聚合酶（Pol），可以绕过各种DNA损伤。人DNA聚合酶κ（hPolκ）是DNA Pols的Y家族成员，是大肠杆菌中DinB的直接对应物。 hPolκ的特点是无法绕过几种DNA加合物[例如，苯并[a] py二醇环氧-N2n-脱氧鸟嘌呤（BPDE-N2n-dG）n和胸腺嘧啶二醇]，并有效地延伸末端不匹配的引物。 hPolκ在结构上与大肠杆菌DinB不同，因为它在N端具有约100个氨基酸的延伸。在这里，据报道，酪氨酸112（Y112）是hPolκ的空间门氨基酸，它通过检测进入核苷酸的20n-羟基来区分dNTP和rNTPsn，在与错配引物末端的延伸反应中起着至关重要的作用。当Y112被丙氨酸替代时，氨基酸变化严重降低了延伸错配引物的催化常数，即kcat，并且使该过程的效率（即kcat / Km）与野生状态相比降低了约400倍。型酶。相反，氨基酸的取代并没有降低与BPDE-N2n-dG相对的dCMP在模板DNA中的插入效率，而它影响hPolκ与BPDE-N2n-dG / dCMP牢固结合于模板引物DNA的能力。我们认为hPolκ的空间门是一个主要的保真度因子，它调节着错配的引物末端的延伸反应。

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