Single-molecule approaches for DNA damage detection and repair: A focus on Repair Assisted Damage Detection (RADD).
Single-molecule approaches for DNA damage detection and repair: A focus on Repair Assisted Damage Detection (RADD).
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DOI：
10.1016/j.dnarep.2023.103533
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发表时间：
2023-09
期刊：
影响因子：
3.8
通讯作者：
Ebenstein, Yuval
中科院分区：
文献类型：
--
作者：
Zur, Tahir Detinis;Deek, Jasline;Ebenstein, Yuval
关键词：
人类基因组不断暴露于各种压力源，这可能导致DNA损伤，突变和疾病。在不同类型的DNA损伤中，单链损伤通常由外部应激和代谢过程引起。DNA损伤的准确检测和定量对于理解修复机制、评估环境影响和评估对治疗的反应至关重要。然而，传统技术在灵敏度和检测多种类型损伤的能力方面存在局限性。近年来，单分子荧光方法已成为精确定位和定量DNA损伤的有力工具。修复辅助损伤检测（RADD）是一种单分子技术，采用特定的修复酶来切除受损的碱基，并结合荧光标记的核苷酸来可视化损伤。这项技术为修复效率和序列特异性损伤提供了有价值的见解。在这篇综述中，我们讨论了RADD检测的原理和应用，突出了它们对增强我们对DNA损伤和修复过程的理解的潜力。单链DNA损伤是最常见但研究最少的DNA损伤形式。单分子荧光检测通过沿延伸的DNA分子对单个损伤沿着计数，提供了最终的损伤检测灵敏度。用于DNA损伤检测的修复酶鸡尾酒可以定制以标记特定的损伤病变或组合用于广谱检测。随着时间的推移跟踪损伤位点的数量为细胞修复动力学提供了有价值的见解。通过结合RADD和光学基因组定位方法，可以将DNA损伤定位到特定的基因组位点。
The human genome is continually exposed to various stressors, which can result in DNA damage, mutations, and diseases. Among the different types of DNA damage, single-strand lesions are commonly induced by external stressors and metabolic processes. Accurate detection and quantification of DNA damage are crucial for understanding repair mechanisms, assessing environmental impacts, and evaluating response to therapy. However, traditional techniques have limitations in sensitivity and the ability to detect multiple types of damage. In recent years, single-molecule fluorescence approaches have emerged as powerful tools for precisely localizing and quantifying DNA damage. Repair Assisted Damage Detection (RADD) is a single-molecule technique that employs specific repair enzymes to excise damaged bases and incorporates fluorescently labeled nucleotides to visualize the damage. This technique provides valuable insights into repair efficiency and sequence-specific damage. In this review, we discuss the principles and applications of RADD assays, highlighting their potential for enhancing our understanding of DNA damage and repair processes. Single-strand DNA damage is the most common yet the least explored form of DNA damage. Single-molecule fluorescence assays provide the ultimate sensitivity for damage detection by counting individual lesions along extended DNA molecules. Repair enzyme cocktails used for DNA damage detection may be tailored to label specific damage lesions or combined for broad spectrum detection. Tracking the number of damage sites over time provides valuable insights into cell repair dynamics. DNA damage may be mapped to specific genomic loci by combining RADD with optical genome mapping approaches.
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