Effect of sequence context on Polζ-dependent error-prone extension past (6-4) photoproducts
Effect of sequence context on Polζ-dependent error-prone extension past (6-4) photoproducts
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DOI：
10.1016/j.dnarep.2019.102771
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发表时间：
2020-03-01
期刊：
影响因子：
3.8
通讯作者：
Hanaoka, Fumio
中科院分区：
文献类型：
--
作者：
Akagi, Jun-ichi;Hashimoto, Keiji;Hanaoka, Fumio
（6-4）嘧啶-嘧啶酮光产物[(6-4)PP]是紫外线辐射引起的主要DNA损伤。（6-4）通过位点特异性翻译DNA合成（TLS）试验可以观察到，PP诱导与下游碱基相反的复杂突变，除了相反的3‘或5’碱基。这些突变发生的机制尚不清楚。为了阐明（6-4）PP诱导突变的机制，我们使用带有位点特异性T（胸腺嘧啶）-T (6-4)PP的复制载体进行了细胞内TLS检测。Rev3(-/-) p53（-/-）小鼠胚胎成纤维细胞（MEF）细胞（Poll缺陷）在绕过T-T (6-4)PP方面几乎完全缺陷，而Revl（-/-）和Poih(-/-) Poli(-/-) Polk(-/-) MEF细胞（Pol eta、Pol iota和Pol kappa缺陷）的绕过活性与野生型细胞相当，这表明y家族TLS聚合酶在绕过活性中是不可缺少的，而Pol zeta可能在延伸阶段起着至关重要的作用。在所有被测试的细胞中，错误掺入最常发生在病变部位（+1位置）之外，这表明Pol zeta依赖的延伸步骤对（6-4）pp诱导的突变至关重要。然后，我们使用一系列位于病变+1或-1位置的不同序列的T-T (6-4)PP模板，研究了序列背景对T-T (6-4)PP旁路的影响，发现T-T (6-4)PP旁路对Pol zeta的依赖性不是序列特异性的。然而，在这些模板中，+1位置的错误合并频率有显著差异。当嘌呤碱基位于-1位置时，A在+1位置的错误结合经常发生。这些结果表明，在（6-4）pp诱变过程中，Pol zeta依赖延伸在诱导碱基替换中起主要作用，其保真度受到病变周围序列背景的影响。
The (6-4) pyrimidine-pyrimidone photoproduct [(6-4)PP] is a major DNA lesion induced by ultraviolet radiation. (6-4)PP induces complex mutations opposite its downstream bases, in addition to opposite 3' or 5' base, as has been observed through a site-specific translesion DNA synthesis (TLS) assay. The mechanism by which these mutations occur is not well understood. To elucidate the mechanisms underlying mutagenesis induced by (6-4) PP, we performed an intracellular TLS assay using a replicative vector with site-specific T(thymidine)-T (6-4)PP. Rev3(-/-) p53(-/-) mouse embryonic fibroblast (MEF) cells (defective in Poll;) were almost completely defective in bypassing T-T (6-4)PP, whereas both Revl(-/-) and Poih(-/-) Poli(-/-) Polk(-/-) MEF cells (defective in Pol eta, Pol iota, and Pol kappa) presented bypassing activity comparable to that of wild-type cells, indicating that Y-family TLS polymerases are dispensable for bypassing activity, whereas Pol zeta plays an essential role, probably at the extension step. Among all cells tested, misincorporation occurred most frequently just beyond the lesion (position +1), indicating that the Pol zeta-dependent extension step is crucial for (6-4)PP-induced mutagenesis. We then examined the effects of sequence context on T-T (6-4)PP bypass using a series of T-T (6-4)PP templates with different sequences at position +1 or -1 to the lesion, and found that the dependency of T-T (6-4)PP bypass on Pol zeta is not sequence specific. However, the misincorporation frequency at position +1 differed significantly among these templates. The misincorporation of A at position +1 occurred frequently when a purine base was located at position -1. These results indicate that Pol zeta-dependent extension plays a major role in inducing base substitutions in (6-4)PP-induced mutagenesis, and its fidelity is affected by sequence context surrounding a lesion.