Background

Genomic instability is one of the enabling characteristics leading to tumour development (Hanahan and Weinberg, 2011). Many sources of DNA damage, either endogenous or exogenous, induce various DNA lesions including double strand breaks (DBSs), which activate the DNA damage response (DDR)–the cell process aimed at preserving DNA integrity. Two main repair pathways are involved in the DSBs repair: non-homologous end joining (NHEJ) and homologous recombination (HR) (Mao et al., 2008). The HR process prevents the loss of genetic information upon DNA damage through a faithful repair using a complementary DNA sequence. By inhibiting the error prone NHEJ and triggering homology-directed DSB repair, DNA end resection has a crucial function in directing the repair pathway choice towards a faithful repair. So, to promote a correct HR, the DSBs are processed through the DNA end-resection machinery which is necessary to generate the long 3′ single-strand DNA (ssDNA) tails essential for the homologous strand invasion. DNA end-resection is a finely regulated process; the first step consists of the recruitment of the MRN complex (MRE11-RAD50-NBSI) and CtIP (RBBP8) onto the DNA lesions to produce short stretches of ssDNA (Stracker and Petrini, 2011) followed by further end-resection mediated by exonuclease I (EXOI) or DNA replication helicase/nuclease 2 (DNA2) in complex with the Bloom syndrome helicases (BLM) (Nimonkar et al., 2011). Subsequently, the replication protein A (RPA) complex binds the ssDNA generated by DNA end-resection, preventing the formation of DNA hairpins (Chen et al., 2013) and to facilitate the loading of RAD51 required for the strand exchange process (Krogh and Symington, 2004).

The evaluation of DNA end-resection is crucial to dissect the molecular mechanisms underlying this finely tuned process and to identify the key players involved. Various methods can be used to assess the DNA end-resection process indirectly, analyzing phosphorylated S4/8 RPA32 as mentioned above, or RAD51 foci, or detection of BrdU in ssDNA following long incubation time (Tkáč et al., 2016). A method to determine the length of resection and the extent of ssDNA at a specific DSB site was developed in 2014 by Tania Paull, although this is based on the expression of an endonuclease (such as AsiSI) to induce DSBs at specific sites within the genome, which is then followed by PCR analysis (Zhou et al., 2014).

The SMART assay was first developed by Huertas and co-authors as a reliable method to measure the length of resected DNA following exposure to any damaging agent in any cellular system, at the level of single molecules (Cruz-García et al., 2014). The assay was based on the previously developed DNA combing assay, which enables the physical stretching of DNA fibres onto a solid support allowing visualization through immunofluorescence (Alfano et al., 2016 and 2017). We recently identified the RNA binding protein HNRNPD as a new player in the HR process and used the SMART assay, along with other techniques, to evaluate DNA end-resection (Alfano et al., 2019). We pulse-labeled HeLa cells with the halogenated IdU (5-Iodo-2′-deoxyuridine)–although any other pyrimidine analogue can be used, such as BrdU (Bromodeoxyuridine)or CldU (5-Chloro-2'-deoxyuridine)–which is incorporated during S phase into the newly synthesized DNA, for approximately 24 h followed by treatment with the DNA damaging agent, camptothecin. At the end of drug incubation time, cells were lysed and the DNA fibres were extracted in non-DNA denaturing conditions (avoiding hydrochloric acid treatment); this is an important criterion for the assay, because the anti-BrdU antibody is able to recognize the DNA-incorporated IdU only when DNA is in the single strand conformation, as is the end-resected DNA. Through the SMART assay, it is possible to visualize the long 3′ single-strand DNA tails and measure the DNA tracts evaluating the efficiency of the endogenous resection machinery without genetic manipulations. Finally, this technique can be used in any model cellular system from bacteria to humans.

The DNA fibres, stretched on a microscope slide, and challenged with the anti-BrdU Ab and the secondary fluorescinated Ab were then visualized through immunofluorescence microscopy. Analysis through the SMART assay allowed us to quantify the amount of resected DNA: by using an image analysis software (Photoshop CS5), we were able to measure the length of the DNA fibre tracts analysing the efficiency of the DNA end-resection machinery.