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2020-08-05
[Abstract] 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 ...
[摘要] [摘要] 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)进行修复。HR过程的一个关键步骤是通过DNA末端切除,产生一个长的3′单链DNA链,这是在DNA链入侵后,以同源区域为模板进行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DNA的可靠修复。已建立的评价DNA末端切除过程的方法是免疫荧光法分析磷酸化S4/8rpa32蛋白病灶(DNA末端切除的标志物)或磷酸化S4/8rpa32蛋白水平。近年来,切除轨迹单分子分析(SMART)被认为是一种可靠的方法,可以通过免疫荧光法观察S期特异性DNA损伤剂(如喜树碱)处理细胞后产生的长3′单链DNA尾。然后,通过图像分析软件(如Photoshop)测量DNA束长度,评价DNA末端切除机的处理能力。DNA纤维的制备是在非变性条件下进行的,因此免疫荧光只检测DSB处理产生的特定的3′单链DNA尾。
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