{"title":"链特异性 PCR 竞争性复制和加合物旁路测定法,用于评估 DNA 加合物如何干扰哺乳动物细胞中的 DNA 复制。","authors":"Jun Yuan, Chen Wang, Xiaomei He, Yinsheng Wang","doi":"10.1016/bs.mie.2024.07.013","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"705 ","pages":"251-270"},"PeriodicalIF":0.0000,"publicationDate":"2024-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Strand-specific PCR-competitive replication and adduct bypass assay for assessing how DNA adducts perturb DNA replication in mammalian cells.\",\"authors\":\"Jun Yuan, Chen Wang, Xiaomei He, Yinsheng Wang\",\"doi\":\"10.1016/bs.mie.2024.07.013\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":18662,\"journal\":{\"name\":\"Methods in enzymology\",\"volume\":\"705 \",\"pages\":\"251-270\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Methods in enzymology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1016/bs.mie.2024.07.013\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/8/10 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q3\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods in enzymology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1016/bs.mie.2024.07.013","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/8/10 0:00:00","PubModel":"Epub","JCR":"Q3","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
引用次数: 0
摘要
人类基因组容易受到各种内源性和外源性因子的损伤。如果不进行修复,由此产生的 DNA 损伤有可能导致突变、基因组不稳定和细胞死亡。虽然现有的体外实验可以利用纯化的转座子合成(TLS)DNA 聚合酶鉴定复制结果,但这类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)检测方法,旨在定量研究哺乳动物细胞中 DNA 病变对复制效率和保真度的影响。结合基因操作,这种方法有助于揭示 TLS 聚合酶在跨 DNA 病变复制中的各种功能。
Strand-specific PCR-competitive replication and adduct bypass assay for assessing how DNA adducts perturb DNA replication in mammalian cells.
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.
期刊介绍:
The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.