Homologous recombination (HR) is necessary to counteract DNA replication stress. Common fragile site (CFS) loci are particularly sensitive to replication stress and undergo pathological ...rearrangements in tumors. At these loci, replication stress frequently activates DNA repair synthesis in mitosis. This mitotic DNA synthesis, termed MiDAS, requires the MUS81-EME1 endonuclease and a non-catalytic subunit of the Pol-delta complex, POLD3. Here, we examine the contribution of HR factors in promoting MiDAS in human cells. We report that RAD51 and BRCA2 are dispensable for MiDAS but are required to counteract replication stress at CFS loci during S-phase. In contrast, MiDAS is RAD52 dependent, and RAD52 is required for the timely recruitment of MUS81 and POLD3 to CFSs in early mitosis. Our results provide further mechanistic insight into MiDAS and define a specific function for human RAD52. Furthermore, selective inhibition of MiDAS may comprise a potential therapeutic strategy to sensitize cancer cells undergoing replicative stress.
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•RAD52 promotes mitotic DNA synthesis (MiDAS) following replication stress•RAD52 promotes recruitment of MUS81 and POLD3 in mitosis•RAD52 deficiency increases mitotic chromosome missegregation•Mitotic DNA synthesis occurs independently of ATR, BRCA2, and RAD51
Tumorigenesis is frequently driven by oncogene activation, which generates so-called DNA replication “stress” and instability at specific genomic loci, called common fragile sites. Bhowmick et al. show that DNA repair synthesis at fragile sites occurs in mitosis and define a role for the homologous recombination factor RAD52 in this process.
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.
The faithful segregation of eukaryotic chromosomes in mitosis requires that the genome be duplicated completely prior to anaphase. However, cells with large genomes sometimes fail to complete ...replication during interphase and instead enter mitosis with regions of incompletely replicated DNA. These regions are processed in early mitosis via a process known as mitotic DNA repair synthesis (MiDAS), but little is known about how cells switch from conventional DNA replication to MiDAS. Using the early embryo of the nematode
as a model system, we show that the TRAIP ubiquitin ligase drives replisome disassembly in response to incomplete DNA replication, thereby providing access to replication forks for other factors. Moreover, TRAIP is essential for MiDAS in human cells, and is important in both systems to prevent mitotic segregation errors. Our data indicate that TRAIP is a master regulator of the processing of incomplete DNA replication during mitosis in metazoa.
Abscission is the final step of cell division when the cytokinetic furrow ingresses completely, leading to midbody formation and plasma membrane fission 1. In human cells, the Aurora-B-driven ...abscission checkpoint delays cytokinesis until any residual chromatin spanning the midbody is removed 2–5. If this does not occur efficiently, uneven segregation of daughter genomes can occur. The mechanism by which the abscission checkpoint becomes satisfied to permit cytokinesis is poorly defined. Here, we identify RIF1 and its binding partner, protein phosphatase 1 (PP1), as being critical for regulation of abscission timing in human cells. We show that RIF1 promotes cytokinesis through recruitment of PP1 to the midbody, which then counteracts Aurora B kinase activity, leading to dephosphorylation of a regulator of abscission timing, CHMP4C 6–10. Although RIF1 binds to unresolved DNA bridges that persist into telophase 11, we show that this cytokinetic function of the RIF1-PP1 axis is not limited to instances where cell division is perturbed by the presence of bridges. Nevertheless, we show that altering the balance of the opposing Aurora B kinase and PP1 phosphatase activities makes cells unresponsive to DNA bridges and sensitizes cells to agents that induce bridge formation. Our data define a new mechanism for regulation of abscission timing and emphasize how antagonism between kinases and phosphatases is a widespread mechanism for determining the timing of mitotic transactions. Because cancer cells experiencing oncogene-induced replication stress generate excessive mitotic DNA bridging 12, targeting this new regulatory pathway could be a promising therapeutic strategy.
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•Ultrafine anaphase bridges (UFBs) activate the abscission checkpoint in human cells•The UFB binding protein RIF1 targets protein phosphatase 1 (PP1) to the midbody•PP1 counteracts the Aurora-B-kinase-driven checkpoint by targeting CHMP4C•The RIF1/PP1 axis regulates abscission timing
Bhowmick et al. have uncovered the mechanism by which the Aurora-B-kinase-driven abscission checkpoint is satisfied to allow cytokinesis in human cells. The DNA repair protein RIF1 localizes to and targets protein phosphatase 1 (PP1) to the midbody. PP1 then counteracts Aurora B to promote abscission.
Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and ...telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.
Oncogene activation creates DNA replication stress (RS) in cancer cells, which can generate under-replicated DNA regions (UDRs) that persist until cells enter mitosis. UDRs also have the potential to ...generate DNA bridges in anaphase cells or micronuclei in the daughter cells, which could promote genomic instability. To suppress such damaging changes to the genome, human cells have developed a strategy to conduct 'unscheduled' DNA synthesis in mitosis (termed MiDAS) that serves to rescue under-replicated loci. Previous studies have shown that MiDAS proceeds via a POLD3-dependent pathway that shows some features of break-induced replication. Here, we define how human cells utilize both DNA gap filling (REV1 and Pol ζ) and replicative (Pol δ) DNA polymerases to complete genome duplication following a perturbed S-phase. We present evidence for the existence of a polymerase-switch during MiDAS that is required for new DNA synthesis at UDRs. Moreover, we reveal that, upon oncogene activation, cancer cell survival is significantly compromised when REV1 is depleted, suggesting that REV1 inhibition might be a feasible approach for the treatment of some human cancers.
DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can ...lead to the dissociation of the replisome and the formation of DNA damage. To model the events stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8-9 complex, a paralog of the MCM2-7 replicative helicase. We show that MCM8-9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8-9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8-9 as an alternative replicative helicase.
An unusual feature of many eukaryotic genomes is the presence of regions that appear intrinsically difficult to copy during the process of DNA replication. Curiously, the location of these ...difficult-to-replicate regions is often conserved between species, implying a valuable role in some aspect of genome organization or maintenance. The most prominent class of these regions in mammalian cells is defined as chromosome fragile sites, which acquired their name because of a propensity to form visible gaps/breaks on otherwise-condensed chromosomes in mitosis. This fragility is particularly apparent following perturbation of DNA replication-a phenomenon often referred to as "replication stress". Here, we review recent data on the molecular basis for chromosome fragility and the role of fragile sites in the etiology of cancer. In particular, we highlight how studies on fragile sites have provided unexpected insights into how the DNA repair machinery assists in the completion of DNA replication.
In this report, the interaction between a phenanthrene–pyrene-based fluorescent probe (PPI) and bovine serum albumin (BSA), a transport protein, has been explored by steady-state emission ...spectroscopy, fluorescence anisotropy, far-ultraviolet circular dichroism (CD), time-resolved spectral measurements, and molecular docking simulation study. The blue shift along with emission enhancement indicates the interaction between PPI and BSA. The binding of the probe causes quenching of BSA fluorescence through both static and dynamic quenching mechanisms, revealing a 1:1 interaction, as delineated from Benesi–Hildebrand plot, with a binding constant of ∼105 M–1, which is in excellent agreement with the binding constant extracted from fluorescence anisotropy measurements. The thermodynamic parameters, ΔH°, ΔS°, and ΔG°, as determined from van’t Hoff relationship indicate the predominance of van der Waals/extensive hydrogen-bonding interactions for the binding phenomenon. The molecular docking and site-selective binding studies reveal the predominant binding of PPI in subdomain IIA of BSA. From the fluorescence resonance energy transfer study, the average distance between tryptophan 213 of the BSA donor and the PPI acceptor is found to be 3.04 nm. CD study demonstrates the reduction of α-helical content of BSA protein on binding with PPI, clearly indicating the change of conformation of BSA.
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