Division of Pathology, National Institute of Health Sciences, Tonomachi, Kawasaki-shi, Kawasaki-ku, Kanagawa, Japan.
Division of Genetics and Mutagenesis, National Institute of Health Sciences, Tonomachi, Kawasaki-shi, Kawasaki-ku, Kanagawa, Japan.
Mutagenesis. 2021 May 31;36(2):155-164. doi: 10.1093/mutage/geab007.
DNA polymerase zeta (Polζ) is a heterotetramer composed of the catalytic subunit Rev3l, Rev7 and two subunits of Polδ (PolD2/Pol31 and PolD3/Pol32), and this polymerase exerts translesion DNA synthesis (TLS) in yeast. Because Rev3l knockout results in embryonic lethality in mice, the functions of Polζ need further investigation in vivo. Then, we noted the two facts that substitution of leucine 979 of yeast Rev3l with methionine reduces Polζ replication fidelity and that reporter gene transgenic rodents are able to provide the detailed mutation status. Here, we established gpt delta mouse knocked in the constructed gene encoding methionine instead of leucine at residue 2610 of Rev3l (Rev3l L2610M gpt delta mice), to clarify the role of Polζ in TLS of chemical-induced bulky DNA adducts in vivo. Eight-week-old gpt delta mice and Rev3l L2610M gpt delta mice were treated with benzo[a]pyrene (BaP) at 0, 40, 80, or 160 mg/kg via single intraperitoneal injection. At necropsy 31 days after treatment, lungs were collected for reporter gene mutation assays. Although the gpt mutant frequency was significantly increased by BaP in both mouse genotypes, it was three times higher in Rev3l L2610M gpt delta than gpt delta mice after treatment with 160 mg/kg BaP. The frequencies of G:C base substitutions and characteristic complex mutations were significantly increased in Rev3l L2610M gpt delta mice compared with gpt delta mice. The BaP dose-response relationship suggested that Polζ plays a central role in TLS when protective mechanisms against BaP mutagenesis, such as error-free TLS, are saturated. Overall, Polζ may incorporate incorrect nucleotides at the sites opposite to BaP-modified guanines and extend short DNA sequences from the resultant terminal mismatches only when DNA is heavily damaged.
DNA 聚合酶 ζ（Polζ）是一种由催化亚基 Rev3l、Rev7 和 Polδ 的两个亚基（PolD2/Pol31 和 PolD3/Pol32）组成的杂四聚体，该聚合酶在酵母中发挥跨损伤 DNA 合成（TLS）的作用。由于 Rev3l 敲除会导致小鼠胚胎致死，因此需要在体内进一步研究 Polζ 的功能。然后，我们注意到两个事实，即酵母 Rev3l 的亮氨酸 979 被蛋氨酸取代会降低 Polζ 的复制保真度，以及报告基因转基因啮齿动物能够提供详细的突变状态。在这里，我们建立了 gpt delta 小鼠，该小鼠在 Rev3l 的残基 2610 处用蛋氨酸取代亮氨酸，构建了编码该基因的基因（Rev3l L2610M gpt delta 小鼠），以阐明 Polζ 在体内化学诱导的大体积 DNA 加合物的 TLS 中的作用。将 8 周龄的 gpt delta 小鼠和 Rev3l L2610M gpt delta 小鼠用苯并[a]芘（BaP）以 0、40、80 或 160mg/kg 的剂量通过单次腹腔注射处理。在处理后 31 天解剖时，收集肺进行报告基因突变测定。尽管在两种小鼠基因型中，BaP 均显著增加了 gpt 突变频率，但在 160mg/kg BaP 处理后，Rev3l L2610M gpt delta 小鼠中的 gpt 突变频率是 gpt delta 小鼠的三倍。Rev3l L2610M gpt delta 小鼠中的 G:C 碱基替换和特征性复杂突变的频率明显高于 gpt delta 小鼠。BaP 剂量反应关系表明，当针对 BaP 致突变性的保护机制（如无差错 TLS）饱和时，Polζ 在 TLS 中发挥核心作用。总体而言，只有在 DNA 受到严重损伤时，Polζ 可能会在与 BaP 修饰的鸟嘌呤相对的位置掺入错误的核苷酸，并从所得的末端错配延伸短的 DNA 序列。
