CFSs are recognized as a driver of genome
CFSs are recognized as a driver of genome instability in human cells and are hotspots for deletions or translocations in cancers (Richards, 2001). The generally accepted model for their expression is that the CFS locus shows a delay in chromatin condensation in early mitosis caused by the persistence of a region of underreplicated DNA (El Achkar, Gerbault-Seureau, Muleris, Dutrillaux, & Debatisse, 2005). This phenomenon is rare in cultured normal cells, but can be exacerbated by the exposure of the cells to agents that partially inhibit DNA synthesis during S-phase (such as aphidicolin; APH) (Glover, Berger, Coyle, & Echo, 1984). One reason why cancer cells seem susceptible to CFS instability is because of the widespread occurrence of oncogene activation in cancer cells, which is a known driver of replication stress (RS) (Hills & Diffley, 2014; Lecona & Fernandez-Capetillo, 2014; Ren et al., 2017).
The mitotic DNA synthesis occurring at CFSs (now termed “MiDAS”) (Bhowmick, Minocherhomji, & Hickson, 2016) was discovered, while we were trying to accurately determine when CFS replication is completed during the Metronidazole in the presence of RS. It had been shown previously that replication was still occurring after the completion of S-phase (Bergoglio et al., 2013; Pedersen, Kruse, Nilsson, Oestergaard, & Lisby, 2015). In our previous studies, the dTTP analogue 5-ethynyl-2′-deoxyuridine (EdU) was utilized to mark sites where DNA replication was still occurring. Because labeling with EdU takes 20–30min, any EdU found in mitotic cells was assumed to have been incorporated into the genome in G2. The use of the CDK1 inhibitor, RO3306 (Vassilev, 2006), to arrest cells at the G2/M boundary allowed us to separate G2 from M-phase, and therefore to add EdU only after cells had entered the prophase of mitosis. Under these circumstances, we could detect EdU on metaphase chromosomes and demonstrate that DNA synthesis was still occurring in early mitosis. Importantly, we could prove that the sites of MiDAS correspond to broken CFS loci by combining EdU detection with fluorescent in situ hybridization (FISH) (Minocherhomji et al., 2015). Moreover, we observed that the entry into prophase triggers the recruitment of DNA structure-specific endonuclease, MUS81–EME1, to CFSs, and that the nuclease action of MUS81–EME1 promotes POLD3-dependent DNA synthesis at CFSs. Although not characterized in detail, our results suggest that MiDAS is a DNA repair-based “salvage” pathway based on a form of BIR that helps cells to complete DNA synthesis at difficult-to-replication loci-like CFSs when they encountered RS. If MiDAS fails to occur, then chromosome missegregation and nondisjunction ensues, particularly in cancer cells with intrinsically high levels of chromosome instability (CIN+ve) (Minocherhomji et al., 2015).
Method The method is largely divided into two parts: see 3.1 To Analyze MiDAS in Prometaphase, 3.2 To Analyze EdU on Metaphase Chromosome Spreads. In each part, the experimental steps are grouped into four categories: (i) to induce RS and arrest cells in G2 phase; (ii) to label cells with EdU in prometaphase or metaphase; (iii) to harvest and fix cells in prometaphase or metaphase for various analysis (i.e., EdU detection, EdU combined with IF, or EdU combined with FISH); and (iv) the detection of the various signals. The experimental flow and the representative images from each type of analysis are illustrated in Fig. 1, Fig. 2.
Conclusions Since its discovery in 2015 as a putative “salvage” pathway for protecting vulnerable regions of the human genome from potentially being missegregated to the daughter cells in mitosis, MiDAS has emerged as a very useful marker denoting those chromosomal sites where RS has been encountered during S-phase. When combined with other methods such as FISH or IF, the detection of MiDAS is a powerful tool not only to define specific loci susceptible to RS, but also to discover which proteins are recruited to loci undergoing MiDAS. Although MiDAS analysis has widespread applicability, it is particularly relevant to studies on cancer cells possessing intrinsic RS driven by oncogene activation. In this regard, a goal of our current studies is to identify a protein involved in MiDAS that might serve as a biomarker for indicating the degree of RS in tumor biopsy specimens, with the ultimate aim of improving the clinical management of cancer patients (Ren et al., 2017).