New England Biolabs, Ipswich, Massachusetts, USA.
Curr Protoc. 2022 Nov;2(11):e595. doi: 10.1002/cpz1.595.
The formation and persistence of DNA damage can impact biological processes such as DNA replication and transcription. To maintain genome stability and integrity, organisms rely on robust DNA damage repair pathways. Techniques to detect and locate DNA damage sites across a genome enable an understanding of the consequences of DNA damage as well as how damage is repaired, which can have key diagnostic and therapeutic implications. Importantly, advancements in technology have enabled the development of high-throughput sequencing-based DNA damage detection methods. These methods require DNA enrichment or amplification steps that limit the ability to quantitate the DNA damage sites. Further, each of these methods is typically tailored to detect only a specific type of damage. RAre DAmage and Repair (RADAR) sequencing is a DNA sequencing workflow that overcomes these limitations and enables detection and quantitation of DNA damage sites in any organism on a genome-wide scale. RADAR-seq works by replacing DNA damage sites with a patch of modified bases that can be directly detected by Pacific Biosciences Single-Molecule Real Time sequencing. Here, we present three protocols that enable detection of thymine dimers and ribonucleotides in bacterial and archaeal genomes. Basic Protocol 1 enables construction of a reference genome required for RADAR-seq analyses. Basic Protocol 2 describes how to locate, quantitate, and compare thymine dimer levels in Escherichia coli exposed to varying amounts of UV light. Basic Protocol 3 describes how to locate, quantitate, and compare ribonucleotide levels in wild-type and ΔRNaseH2 Thermococcus kodakarensis. Importantly, all three protocols provide in-depth steps for data analysis. Together they serve as proof-of-principle experiments that will allow users to adapt the protocols to locate and quantitate a wide variety of DNA damage sites in any organism. © 2022 New England Biolabs. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Constructing a reference genome utilizing SMRT sequencing Basic Protocol 2: Mapping and quantitating genomic thymine dimer formation in untreated versus UV-irradiated E. coli using RADAR-seq Basic Protocol 3: Mapping and quantitating genomic ribonucleotide incorporation in wildtype versus ΔRNaseH2 T. kodakarensis using RADAR-seq.
DNA 损伤的形成和持续存在会影响生物过程，如 DNA 复制和转录。为了维持基因组的稳定性和完整性，生物体依赖于强大的 DNA 损伤修复途径。检测和定位基因组中 DNA 损伤位点的技术可以帮助我们了解 DNA 损伤的后果以及损伤是如何修复的，这可能具有关键的诊断和治疗意义。重要的是，技术的进步使高通量测序的 DNA 损伤检测方法得以发展。这些方法需要进行 DNA 富集或扩增步骤，这限制了定量分析 DNA 损伤位点的能力。此外，这些方法中的每一种通常都专门用于检测特定类型的损伤。RADAR 测序是一种 DNA 测序工作流程，它克服了这些限制，能够在全基因组范围内检测和定量分析任何生物体的 DNA 损伤位点。RADAR-seq 通过用修饰碱基替换 DNA 损伤位点来工作，这些修饰碱基可以通过 Pacific Biosciences 的单分子实时测序直接检测到。在这里，我们提出了三个协议，可用于检测细菌和古菌基因组中的胸腺嘧啶二聚体和核糖核苷酸。基本方案 1 可构建 RADAR-seq 分析所需的参考基因组。基本方案 2 描述了如何在不同剂量的紫外光下定位、定量和比较暴露于紫外光的大肠杆菌中的胸腺嘧啶二聚体水平。基本方案 3 描述了如何在野生型和 ΔRNaseH2Thermococcus kodakarensis 中定位、定量和比较核糖核苷酸水平。重要的是，所有三个方案都提供了深入的数据分析步骤。它们共同作为原理验证实验，使用户能够适应这些方案来定位和定量分析任何生物体中的各种 DNA 损伤位点。© 2022 New England Biolabs。Wiley Periodicals LLC 出版的当前方案。基本方案 1：利用 SMRT 测序构建参考基因组基本方案 2：利用 RADAR-seq 定位和定量分析未经处理和经紫外线照射的大肠杆菌中的基因组胸腺嘧啶二聚体形成基本方案 3：利用 RADAR-seq 定位和定量分析野生型和 ΔRNaseH2Thermococcus kodakarensis 中的基因组核糖核苷酸掺入。
