Department of Chemistry, University of California, Riverside, CA, United States.
Department of Chemistry, University of California, Riverside, CA, United States.
Methods Enzymol. 2024;705:251-270. doi: 10.1016/bs.mie.2024.07.013. Epub 2024 Aug 10.
Human genomes are susceptible to damage by a variety of endogenous and exogenous agents. If not repaired, the resulting DNA lesions can potentially lead to mutations, genome instability, and cell death. While existing in vitro experiments allow for characterizing replication outcomes from the use of purified translesion synthesis (TLS) DNA polymerases, such studies often lack the sophistication and dynamic nature of cellular contexts. Here, we present a strand-specific PCR-based Competitive Replication and Adduct Bypass (ssPCR-CRAB) assay designed to investigate quantitatively the impact of DNA lesions on replication efficiency and fidelity in mammalian cells. Combined with genetic manipulation, this approach facilitates the revelation of diverse functions of TLS polymerases in replication across DNA lesions.
人类基因组容易受到各种内源性和外源性因素的损害。如果不加以修复，由此产生的 DNA 损伤可能导致突变、基因组不稳定和细胞死亡。虽然现有的体外实验可以描述使用纯化的跨损伤合成（TLS）DNA 聚合酶的复制结果，但这些研究往往缺乏细胞环境的复杂性和动态性。在这里，我们提出了一种基于链特异性 PCR 的竞争性复制和加合物旁路（ssPCR-CRAB）测定法，旨在定量研究 DNA 损伤对哺乳动物细胞中复制效率和保真度的影响。结合遗传操作，这种方法有助于揭示 TLS 聚合酶在跨 DNA 损伤复制中的多种功能。
