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In Vitro Bypass of Thymidine Glycol by DNA Polymerase θ Forms Sequence-Dependent Frameshift Mutations
Biochemistry ( IF 2.9 ) Pub Date : 2017-12-15 00:00:00 , DOI: 10.1021/acs.biochem.7b01093 Daniel J Laverty 1 , Marc M Greenberg 1
Biochemistry ( IF 2.9 ) Pub Date : 2017-12-15 00:00:00 , DOI: 10.1021/acs.biochem.7b01093 Daniel J Laverty 1 , Marc M Greenberg 1
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Unrepaired DNA lesions block replication and threaten genomic stability. Several specialized translesion polymerases, including polymerase θ (Pol θ), contribute to replicative bypass of these lesions. The role of Pol θ in double-strand break repair is well-understood, but its contribution to translesion synthesis is much less so. We describe the action of Pol θ on templates containing thymidine glycol (Tg), a major cytotoxic, oxidative DNA lesion that blocks DNA replication. Unrepaired Tg lesions are bypassed in human cells by specialized translesion polymerases by one of two distinct pathways: high-fidelity bypass by the combined action of Pol κ and Pol ζ or weakly mutagenic bypass by Pol θ. Here we report that in vitro bypass of Tg by Pol θ results in frameshift mutations (deletions) in a sequence-dependent fashion. Steady-state kinetic analysis indicated that one- and two-nucleotide deletions are formed 9- and 6-fold more efficiently, respectively, than correct, full-length bypass products. Sequencing of in vitro bypass products revealed that bypass preference decreased in the following order on a template where all three outcomes were possible: two-nucleotide deletion > correct bypass > one-nucleotide deletion. These results suggest that bypass of Tg by Pol θ results in mutations opposite the lesion, as well as frameshift mutations.
中文翻译：
DNA 聚合酶 θ 体外旁路胸苷乙二醇形成序列依赖性移码突变
未修复的 DNA 损伤会阻碍复制并威胁基因组的稳定性。几种专门的跨损伤聚合酶，包括聚合酶 θ (Pol θ)，有助于这些损伤的复制旁路。 Pol θ 在双链断裂修复中的作用已广为人知，但其对跨损伤合成的贡献却少之又少。我们描述了 Pol θ 对含有胸苷乙二醇 (Tg) 的模板的作用，胸苷乙二醇是一种主要的细胞毒性、氧化性 DNA 损伤，可阻止 DNA 复制。人体细胞中未修复的 Tg 损伤被专门的跨损伤聚合酶通过两种不同途径之一绕过：通过 Pol κ 和 Pol ζ 的联合作用进行高保真旁路，或通过 Pol θ 进行弱诱变旁路。在这里，我们报告体外通过 Pol θ 绕过 Tg 会导致序列依赖性的移码突变（缺失）。稳态动力学分析表明，形成一核苷酸和二核苷酸缺失的效率分别比正确的全长旁路产物高 9 倍和 6 倍。体外旁路产物的测序表明，在所有三种结果都可能的模板上，旁路偏好按以下顺序下降：两核苷酸删除>正确旁路>一核苷酸删除。这些结果表明，Pol θ 绕过 Tg 会导致与病变相对的突变以及移码突变。
更新日期：2017-12-15
中文翻译：
DNA 聚合酶 θ 体外旁路胸苷乙二醇形成序列依赖性移码突变
未修复的 DNA 损伤会阻碍复制并威胁基因组的稳定性。几种专门的跨损伤聚合酶，包括聚合酶 θ (Pol θ)，有助于这些损伤的复制旁路。 Pol θ 在双链断裂修复中的作用已广为人知，但其对跨损伤合成的贡献却少之又少。我们描述了 Pol θ 对含有胸苷乙二醇 (Tg) 的模板的作用，胸苷乙二醇是一种主要的细胞毒性、氧化性 DNA 损伤，可阻止 DNA 复制。人体细胞中未修复的 Tg 损伤被专门的跨损伤聚合酶通过两种不同途径之一绕过：通过 Pol κ 和 Pol ζ 的联合作用进行高保真旁路，或通过 Pol θ 进行弱诱变旁路。在这里，我们报告体外通过 Pol θ 绕过 Tg 会导致序列依赖性的移码突变（缺失）。稳态动力学分析表明，形成一核苷酸和二核苷酸缺失的效率分别比正确的全长旁路产物高 9 倍和 6 倍。体外旁路产物的测序表明，在所有三种结果都可能的模板上，旁路偏好按以下顺序下降：两核苷酸删除>正确旁路>一核苷酸删除。这些结果表明，Pol θ 绕过 Tg 会导致与病变相对的突变以及移码突变。
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