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

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 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.

Keywords: BrdU; DNA end-resection; DNA repair; Homologous Recombination; Immunofluorescence.

Figure 1.. Schematics of SMART assay.

First of all, label HeLa cells with IdU for 24 h followed by CPT treatment; at the end of incubation trypsinize cells and dilute them at a 2.5 x 10⁵ cells/ml concentration. Mix labelled and unlabelled cells 1:8 and spot 2.5 μl of this mix onto the upper part of the slide; after incubation with 7.5 μl of spreading buffer, tilt the slide form the end to allow the stretching of the DNA fibres.

Figure 2.. Representative images of the SMART technique.

Panel A shows two images from HeLawt or siEXOI cells treated with 1 μM of campthotecin for one hour; following the SMART assay images were analyzed as reported in the protocol through the Fiji software. As shown in panel B the silencing of EXOI, one of the principal exonucleases involved in the generation of single strand DNA upon DNA damage, reduces the length of the processed DNA up to two times. The Figure 3B reports the mean ± standard deviation of the fibre length measured in μm, for each condition.

Figure 3.. A representative image opened with the Fiji software showing in the upper left part the pixel resolution and μm conversion; in the lower corner of the figure the μm dimension.

A. Micrometer conversion into kilobase depends on the DNA type, the nucleosome and chromatin fibre assembly; for HeLa cells and the combing assay 1 μm of DNA fibre length, during the S-phase, should approximately correspond to 2.59 kb as reported originally by Jackson and Pombo (Jackson and Pombo, 1998) and others ( Bianco et al., 2012 ; Jamroskovic et al., 2020 ; Petermann et al., 2008 ). B. A magnification image of DNA fibre showing in red (start) and blue (end) the dimension of the analysed DNA tracts. C. The yellow arrows indicate, in lower part of the figure, the direction and the length of the DNA fibres; these fibres cannot be used for the measurement given the high number of other DNA fibres, which reduce the identification of single DNA traits. The red arrow indicates a fibre at a density and fluorescence, which are optimal for the analysis of the single DNA traits. D. Example of a screenshot of the Straight Line tool from the Fiji software analysis.