Effect of sequence context on Polζ-dependent error-prone extension past (6-4) photoproducts.,DNA Repair

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Effect of sequence context on Polζ-dependent error-prone extension past (6-4) photoproducts.
DNA Repair ( IF 3.0 ) Pub Date : 2019-12-14 , DOI: 10.1016/j.dnarep.2019.102771
Jun-Ichi Akagi ₁ , Keiji Hashimoto ₂ , Kenji Suzuki ₃ , Masayuki Yokoi ₄ , Niels de Wind ₅ , Shigenori Iwai ₆ , Haruo Ohmori ₃ , Masaaki Moriya ₂ , Fumio Hanaoka ₇

Affiliation

Department of Life Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo, 171-8588, Japan; Division of Pathology, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa, 210-9501, Japan.
Laboratory of Chemical Biology, Department of Pharmacological Sciences, State University of New York, Stony Brook, NY, 11794-8651, USA.
Department of Life Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo, 171-8588, Japan.
Department of Life Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo, 171-8588, Japan; Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo, 657-8501, Japan.
Department of Human Genetics, Leiden University Medical Center, 2300 RC, Leiden, the Netherlands.
Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka, 560-8531, Japan.
Department of Life Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo, 171-8588, Japan; Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka, 565-0871, Japan; National Institute of Genetics, 1111 Yata, Mishima, Shizuoka, 411-8540, Japan.

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 Polζ) were almost completely defective in bypassing T-T (6-4)PP, whereas both Rev1-/- and Polh-/-Poli-/-Polk-/- MEF cells (defective in Polη, Polι, and Polκ) presented bypassing activity comparable to that of wild-type cells, indicating that Y-family TLS polymerases are dispensable for bypassing activity, whereas Polζ 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 Polζ-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 Polζ 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 Polζ-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.

中文翻译：

序列上下文对过去（6-4）个光产物的Polζ依赖的易错扩展的影响。

（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）PP诱导了与其下游碱基相对的复杂突变，除了相对的3'或5'碱基外，正如通过位点特异性跨病变DNA合成（TLS）分析所观察到的那样。这些突变发生的机制尚不清楚。为了阐明由（6-4）PP诱导诱变的潜在机制，我们使用具有位点特异性T（胸苷）-T（6-4）PP的复制载体进行了细胞内TLS分析。Rev3-/-p53-/-小鼠胚胎成纤维细胞（MEF）（在Polζ中有缺陷）在绕过TT（6-4）PP时几乎完全有缺陷，而Rev1-/-和Polh-/-Poli-/-Polk -/-MEF细胞（在Polη，Polι和Polκ中有缺陷）呈现出与野生型细胞相当的旁路活性，这表明Y家族TLS聚合酶对于绕过活性是必不可少的，而Polζ可能在延伸步骤中起着至关重要的作用。在所有测试的细胞中，错误掺入最常发生在病变附近（位置+1），这表明Polζ依赖性延伸步骤对于（6-4）PP诱导的诱变至关重要。然后，我们使用一系列在病变位置+1或-1处具有不同序列的TT（6-4）PP模板，检查了序列上下文对TT（6-4）PP旁路的影响，并发现TT的依赖性Polζ上的（6-4）PP旁路不是序列特异性的。但是，在这些模板之间，位置+1处的掺入错误频率显着不同。当嘌呤碱基位于位置-1时，经常会在位置+1发生A的错误掺入。

更新日期：2019-12-17

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