Abstract
DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.
Highlights
The accurate duplication of the genome is fundamental for cell viability
We provide evidence supporting the hypothesis that mitotic DNA synthesis (MiDAS) is mediated by break-induced DNA replication (BIR), as well as mechanistic insights that help explain why common fragile sites (CFSs) are prone to genomic instability in human cancers and in cells treated with DNA replication inhibitors
DNA replication directionality in MiDAS To explore the generality of these observations, we examined a second cell line, HeLa, which is derived from a different tissue type than U2OS cells
Summary
The accurate duplication of the genome is fundamental for cell viability. In eukaryotes, DNA replication initiates at specific genomic locations called replication origins. Origin firing generates two replication forks that advance bidirectionally along the parental DNA template until they each encounter a converging replication fork.[1] in principle, the above process can ensure complete replication of the genome, several obstacles can be encountered that have the potential to arrest or perturb ongoing fork progression. These include atypical structures formed in the DNA template, such as hairpins and G-quadruplexes, or encounters with the machineries carrying out other DNA metabolic processes, such as transcription.[2,3,4,5] Curiously, many eukaryotic genomes harbor evolutionarily conserved regions that appear intrinsically difficult to replicate. The most extensively studied of these regions are the so-called common fragile sites (CFSs).[6,7,8,9,10,11] These sites are of broad interest, because they very frequently correspond to sites of genomic rearrangements in human cancers; six of the ten most common loci for recurrent focal deletions in human cancers lie within CFSs.[12]
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