Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells
Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells
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DOI：
10.1073/pnas.1216894110
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发表时间：
2013-04-16
影响因子：
11.1
通讯作者：
Livneh, Zvi
中科院分区：
文献类型：
--
作者：
Izhar, Lior;Ziv, Omer;Livneh, Zvi
DNA 损伤会阻碍复制叉并导致单链缺口的形成。 DNA 损伤耐受机制可以减轻这些复制并发症，从而防止细胞死亡、基因组不稳定和致癌等有害结果。两种主要的耐受策略是跨损伤 DNA 合成 (TLS)，其中低保真度 DNA 聚合酶绕过阻断损伤，以及同源依赖性修复（HDR；复制后修复），基于同源姐妹染色单体。在这里，我们描述了一种独特的高分辨率方法，用于同时分析哺乳动物基因组中定义的 DNA 损伤的 TLS 和 HDR。该方法基于使用噬菌体整合酶介导的整合，将携带特定位点 DNA 损伤的质粒插入哺乳动物染色体。使用这种方法，我们证明哺乳动物细胞使用 HDR 来耐受其基因组中的 DNA 损伤。此外，对紫外线诱导的 6-4 光产物、烟草烟雾诱导的苯并[a]芘-鸟嘌呤加合物和人工三亚甲基插入物的耐受性分析表明，这三种损伤中的每一种都能被 TLS 和 HDR 耐受。我们还确定了人类基因组中 TLS 期间与这些损伤相对的核苷酸插入的特异性。这种独特的方法将有助于阐明哺乳动物染色体中 DNA 损伤耐受性的机制及其与癌发生等病理过程的联系。
DNA lesions can block replication forks and lead to the formation of single-stranded gaps. These replication complications are mitigated by DNA damage tolerance mechanisms, which prevent deleterious outcomes such as cell death, genomic instability, and carcinogenesis. The two main tolerance strategies are translesion DNA synthesis (TLS), in which low-fidelity DNA polymerases bypass the blocking lesion, and homology-dependent repair (HDR; post-replication repair), which is based on the homologous sister chromatid. Here we describe a unique high-resolution method for the simultaneous analysis of TLS and HDR across defined DNA lesions in mammalian genomes. The method is based on insertion of plasmids carrying defined site-specific DNA lesions into mammalian chromosomes, using phage integrase-mediated integration. Using this method we show that mammalian cells use HDR to tolerate DNA damage in their genome. Moreover, analysis of the tolerance of the UV light-induced 6-4 photoproduct, the tobacco smoke-induced benzo[a]pyrene-guanine adduct, and an artificial trimethylene insert shows that each of these three lesions is tolerated by both TLS and HDR. We also determined the specificity of nucleotide insertion opposite these lesions during TLS in human genomes. This unique method will be useful in elucidating the mechanism of DNA damage tolerance in mammalian chromosomes and their connection to pathological processes such as carcinogenesis.