Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Proc Natl Acad Sci U S A. 2013 Apr 16;110(16):E1462-9. doi: 10.1073/pnas.1216894110. Epub 2013 Mar 25.
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; postreplication 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.
DNA 损伤可阻断复制叉，并导致单链缺口的形成。这些复制相关的复杂问题可通过 DNA 损伤容忍机制得到缓解，该机制可预防诸如细胞死亡、基因组不稳定性和癌变等有害后果。两种主要的容忍策略是跨损伤 DNA 合成（TLS）和同源依赖修复（HDR；复制后修复），前者是指低保真度 DNA 聚合酶绕过阻碍性损伤，后者则基于同源姐妹染色单体。本文中，我们描述了一种独特的高分辨率方法，可同时分析哺乳动物基因组中特定 DNA 损伤处的 TLS 和 HDR。该方法基于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中。我们利用该方法发现，哺乳动物细胞利用 HDR 来容忍其基因组中的 DNA 损伤。此外，对 UV 光诱导的 6-4 光产物、烟草烟雾诱导的苯并[a]芘鸟嘌呤加合物以及人工三烯插入物这三种损伤的耐受性分析表明，这三种损伤均可通过 TLS 和 HDR 进行容忍。我们还确定了在人类基因组中 TLS 过程中这些损伤位点的核苷酸插入特异性。该独特方法将有助于阐明哺乳动物染色体中 DNA 损伤容忍的机制及其与癌变等病理过程的关联。
