Our conventional understanding of the process of DNA replication is that it occurs in the S-phase of the cell division cycle. However, during investigations into the mechanism by which common fragile sites (CFSs) drive genome instability, we observed that some DNA synthesis was still occurring in early mitosis at these loci. This curious phenomenon of mitotic DNA synthesis (which we now term "MiDAS") appears to be a form of break-induced DNA replication (BIR), a DNA repair process based on homologous recombination that has been characterized in detail only in lower eukaryotes. During MiDAS, it is proposed that parts of the human genome that are not fully replicated when cells enter mitotic prophase complete their replicative cycle at that point. To date, the loci that most depend upon this process are those whose replication can be affected by oncogene-induced DNA replication stress (RS), most notably, CFSs. From our studies, it is clear that the successful completion of MiDAS at CFSs can minimize chromosome missegregation and nondisjunction. Nevertheless, it is still not clear which loci that can undergo MiDAS, whether MiDAS is associated with mutations or genome rearrangements, or whether MiDAS really is a form of BIR. In this review, we describe methods for detecting MiDAS both in prometaphase cells and directly on isolated metaphase chromosomes. In addition, we have included methods for combining MiDAS detection either with immunofluorescence (IF) detection of proteins that are recruited to the MiDAS loci, or with fluorescence in situ hybridization using probes that target specific genomic loci.
Keywords: Common fragile sites; DNA replication stress; EdU; Fluorescence in situ hybridization; Immunofluorescence; Mitosis.
© 2018 Elsevier Inc. All rights reserved.