Mutagenicity of a Model DNA-Peptide Cross-Link in Human Cells: Roles of Translesion Synthesis DNA Polymerases.
Mutagenicity of a Model DNA-Peptide Cross-Link in Human Cells: Roles of Translesion Synthesis DNA Polymerases.
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DOI：
10.1021/acs.chemrestox.6b00397
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发表时间：
2017-02-20
影响因子：
4.1
通讯作者：
Basu AK
中科院分区：
文献类型：
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作者：
Pande P;Ji S;Mukherjee S;Schärer OD;Tretyakova NY;Basu AK
当细胞DNA暴露于各种试剂（包括抗肿瘤药物、UV光、过渡金属和活性氧物质）时，形成DNA-蛋白质交联。他们被认为有助于癌症，衰老和神经退行性疾病。已经提出，细胞中形成的DNA-蛋白质交联经受蛋白水解降解为相应的DNA-肽交联（DpC）。为了研究DpCs对DNA复制的影响，我们构建了含有10-mer Myc肽共价连接到7-deaza-dG的C7的质粒DNA，7-deaza-dG是N7-dG病变的水解稳定模拟物。在人胚肾细胞（HEK 293 T）中转染后，回收子代质粒并测序。与未修饰的对照相比，DpC后的转换合成（TLS）为76%。DpC诱导20%的靶向G→A和G→T加上15%的半靶向突变，特别是在病变部位3'的鸟嘌呤（G5）5个碱基处。DpC的蛋白水解消化大大降低了突变频率，表明共价连接的10-mer肽是观察到的突变的原因。在siRNA敲低pol η、pol κ或pol β后，TLS效率和靶向突变降低，表明它们参与DpC病变的易错旁路。然而，G5处的半靶向突变仅在敲低pol后减少，表明其在这种类型的突变中的关键作用。我们的研究结果表明，在鸟嘌呤的N7位置形成的DpCs可以诱导人类细胞中的靶向和半靶向突变，并且TLS聚合酶在其易错旁路中起着关键作用。
DNA-protein cross-links are formed upon exposure of cellular DNA to various agents, including antitumor drugs, UV light, transition metals, and reactive oxygen species. They are thought to contribute to cancer, aging, and neurodegenerative diseases. It has been proposed that DNA-protein cross-links formed in cells are subject to proteolytic degradation to the corresponding DNA-peptide cross-links (DpCs). To investigate the effects of DpCs on DNA replication, we have constructed plasmid DNA containing a 10-mer Myc peptide covalently linked to C7 of 7-deaza-dG, a hydrolytically stable mimic of N7-dG lesions. Following transfection in human embryonic kidney cells (HEK 293T), progeny plasmids were recovered and sequenced. Translesion synthesis (TLS) past DpC was 76% compared to unmodified control. The DpC induced 20% targeted G→A and G→T plus 15% semi-targeted mutations, notably at a guanine (G5) five bases 3’ to the lesion site. Proteolytic digestion of the DpC reduced the mutation frequency considerably, indicating that the covalently attached 10-mer peptide was responsible for the observed mutations. TLS efficiency and targeted mutations were reduced upon siRNA knockdown of pol η, pol κ, or pol ζ, indicating that they participate in error-prone bypass of the DpC lesion. However, the semi-targeted mutation at G5 was only reduced upon knockdown of pol ζ, suggesting its critical role in this type of mutations. Our results indicate that DpCs formed at the N7 position of guanine can induce both targeted and semi-targeted mutations in human cells and that the TLS polymerases play a critical role in their error-prone bypass.