SMART (Single Molecule Analysis of Resection Tracks) Technique for Assessing DNA end-Resection in Response to DNA Damage

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.