SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage
SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage
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DOI：
10.21769/bioprotoc.3701
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发表时间：
2020-08-05
期刊：
影响因子：
0.8
通讯作者：
Alfano, Luigi
中科院分区：
文献类型：
--
作者：
Altieri, Angela;Dell'Aquila, Milena;Alfano, Luigi
DNA双链断裂（DSB）是影响基因组完整性的最具毒性的损伤之一。DSB主要通过非同源末端连接（NHEJ）和同源重组（HR）修复。HR过程的关键步骤是通过DNA末端切除产生长的3'单链DNA片段，这是在DNA链侵入后使用同源区域作为模板引发DNA合成所必需的。DNA末端切除抑制NHEJ并触发同源定向的DSB修复，最终保证忠实的DNA修复。已建立的评价DNA末端切除过程的方法是磷酸-S4/8 RPA 32蛋白灶（DNA末端切除的标志物）的免疫荧光分析，或通过Western印迹法测定磷酸-S4/8 RPA 32蛋白水平。最近，切除轨迹的单分子分析（SMART）已被描述为通过免疫荧光可视化在用S期特异性DNA损伤剂（如喜树碱）处理细胞时产生的长3'单链DNA尾的可靠方法。然后，可以通过图像分析软件（如Photoshop）测量DNA束长度，以评估DNA末端切除机器的持续合成能力。DNA纤维的制备在非变性条件下进行，使得免疫荧光仅检测由DSB处理产生的特异性长3'单链DNA尾。
DNA double strand breaks (DSBs) are among the most toxic lesions affecting genome integrity. DSBs are mainly repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). A crucial step of the HR process is the generation, through DNA end-resection, of a long 3' single-strand DNA stretch, necessary to prime DNA synthesis using a homologous region as a template, following DNA strand invasion. DNA end resection inhibits NHEJ and triggers homology-directed DSB repair, ultimately guaranteeing a faithful DNA repair. Established methods to evaluate the DNA end-resection process are the immunofluorescence analysis of the phospho-S4/8 RPA32 protein foci, a marker of DNA end-resection, or of the phospho-S4/8 RPA32 protein levels by Western blot. Recently, the Single Molecule Analysis of Resection Tracks ( SMART) has been described as a reliable method to visualize, by immunofluorescence, the long 3' single-strand DNA tails generated upon cell treatment with a S-phase specific DNA damaging agent (such as camptothecin). Then, DNA tract lengths can be measured through an image analysis software (such as Photoshop), to evaluate the processivity of the DNA end-resection machinery. The preparation of DNA fibres is performed in non-denaturing conditions so that the immunofluorescence detects only the specific long 3' single-strand DNA tails, generated from DSB processing.