Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University , Max and Anna Web Street, Ramat-Gan, Israel 5290002.
J Am Chem Soc. 2014 May 28;136(21):7771-6. doi: 10.1021/ja503677n. Epub 2014 May 15.
DNA damage and repair are linked to fundamental biological processes such as metabolism, disease, and aging. Single-strand lesions are the most abundant form of DNA damage; however, methods for characterizing these damage lesions are lacking. To avoid double-strand breaks and genomic instability, DNA damage is constantly repaired by efficient enzymatic machinery. We take advantage of this natural process and harness the repair capacity of a bacterial enzymatic cocktail to repair damaged DNA in vitro and incorporate fluorescent nucleotides into damage sites as part of the repair process. We use single-molecule imaging to detect individual damage sites in genomic DNA samples. When the labeled DNA is extended on a microscope slide, damage sites are visualized as fluorescent spots along the DNA contour, and the extent of damage is easily quantified. We demonstrate the ability to quantitatively follow the damage dose response to different damaging agents as well as repair dynamics in response to UV irradiation in several cell types. Finally, we show the modularity of this single-molecule approach by labeling DNA damage in conjunction with 5-hydroxymethylcytosine in genomic DNA extracted from mouse brain tissue.
DNA 损伤与修复与新陈代谢、疾病和衰老等基本生物学过程有关。单链损伤是 DNA 损伤中最常见的形式；然而，目前缺乏对这些损伤的特征描述方法。为了避免双链断裂和基因组不稳定，DNA 损伤会被高效的酶促机制不断修复。我们利用这一自然过程，利用细菌酶混合物的修复能力，在体外修复受损 DNA，并将荧光核苷酸整合到损伤部位，作为修复过程的一部分。我们使用单分子成像技术来检测基因组 DNA 样本中的单个损伤部位。当标记的 DNA 在显微镜载玻片上延伸时，损伤部位会作为荧光点沿着 DNA 轮廓可视化，并且可以轻松定量损伤程度。我们证明了该方法能够定量跟踪不同损伤剂的损伤剂量反应以及对几种细胞类型中紫外线照射的修复动力学。最后，我们通过与从小鼠脑组织中提取的基因组 DNA 中的 5-羟甲基胞嘧啶结合标记 DNA 损伤，展示了这种单分子方法的模块化。
