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Hematopoiesis is particularly sensitive to DNA damage. Myeloid tumor incidence increases in patients with DNA repair defects and after chemotherapy. It is not known why hematopoietic cells are highly vulnerable to DNA damage. Addressing this question is complicated by the paucity of mouse models of hematopoietic malignancies due to defective DNA repair. We show that DNA repair-deficient Mcm8- and Mcm9-knockout mice develop myeloid tumors, phenocopying prevalent myelodysplastic syndromes. We demonstrate that these tumors are preceded by a lifelong DNA damage burden in bone marrow and that they acquire proliferative capacity by suppressing signaling of the tumor suppressor and cell cycle controller RB, as often seen in patients. Finally, we found that absence of MCM9 and the tumor suppressor Tp53 switches tumorigenesis to lymphoid tumors without precedent myeloid malignancy. Our results demonstrate that MCM8/9 deficiency drives myeloid tumor development and establishes a DNA damage burdened mouse model for hematopoietic malignancies., (Copyright © 2019. Published by Elsevier Inc.)

Replication stress is a strong and early driving force for genomic instability and tumor development. Beside replicative DNA polymerases, an emerging group of specialized DNA polymerases is involved in the technical assistance of the replication machinery in order to prevent replicative stress and its deleterious consequences. During S-phase, altered progression of the replication fork by endogenous or exogenous impediments induces replicative stress, causing cells to reach mitosis with genomic regions not fully duplicated. Recently, specific mechanisms to resolve replication intermediates during mitosis with the aim of limiting DNA damage transmission to daughter cells have been identified. In this review, we detail the two major actions of specialized DNA polymerases that limit DNA damage transmission: the prevention of replicative stress by non-B DNA replication and the recovery of stalled replication forks., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Failure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity.

DNA polymerase beta (Pol beta) is an error-prone enzyme which has been found to be overexpressed in several human tumors. By using a couple of recombinant CHO cells differing only from the exogenous expression of Pol beta, we showed here that cells overexpressing Pol beta are much more sensitive to IR treatments by increasing apoptosis. We also found that the surviving cells displayed an hypermutator phenotype which could be explained by different pathways involving Pol beta, such as (i) an increased capacity to incorporate into DNA the mutagenic dGTP analog, 8-oxo-dGTP, one of the most abundant purine-derived nucleotides exposed to gamma-irradiation, (ii) the induction of IR-induced DNA breaks and (iii) accumulation of chromosome aberrations induced by radiation. Alteration of Pol beta expression in irradiated cells thus appears to strengthen both cell death and genetic changes associated with a malignant phenotype. These data provide new insights into the cellular response to radiations and the associated carcinogenic consequences.

Abstract: The cell life span depends on a subtle equilibrium between the accurate duplication of the genomic DNA and less stringent DNA transactions which allow cells to tolerate mutations associated with DNA damage. The physiological role of the alternative, specialized or TLS (translesion synthesis) DNA polymerases could be to favor the necessary “flexibility” of the replication machinery, by allowing DNA replication to occur even in the presence of blocking DNA damage. As these alternative DNA polymerases are inaccurate when replicating undamaged DNA, the regulation of their expression needs to be carefully controlled. Evidence in the literature supports that dysregulation of these error-prone enzymes contributes to the acquisition of a mutator phenotype that, along with defective cell cycle control or other genome stability pathways, could be a motor for accelerated tumor progression. [ABSTRACT FROM AUTHOR]

The major challenge of DNA replication is to provide daughter cells with intact and fully duplicated genetic material. However, various endogenous or environmental factors can slow down or stall DNA replication forks; these replication problems are known to fuel genomic instability and associated pathology, including cancer progression. Whereas the mechanisms emphasizing the source and the cellular responses of replicative problems have attracted much consideration over the past decade, the propagation through mitosis of genome modification and its heritability in daughter cells when the stress is not strong enough to provoke a checkpoint response in G2/M was much less documented. Some recent studies addressing whether low replication stress could impact the DNA replication program of the next generation of cells made the remarkable discovery that DNA damage can indeed be transmitted to daughter cells and can be processed in the subsequent S-phase, and that the replication timing program at a subset of chromosomal domains can also be impacted in the next generation of cells. Such a progression of replication problems into mitosis and daughter cells may appear counter-intuitive, but it could offer considerable advantages by alerting the next generation of cells of potentially risky loci and offering the possibility of an adaptive mechanism to anticipate a reiteration of problems, notably for cancer cells in the context of resistance to therapy. [ABSTRACT FROM AUTHOR]

DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains. [ABSTRACT FROM AUTHOR]

DNA can experience "replication stress", an important source of genome instability, induced by various external or endogenous impediments that slow down or stall DNA synthesis. While genome instability is largely documented to favor both tumor formation and heterogeneity, as well as drug resistance, conversely, excessive instability appears to suppress tumorigenesis and is associated with improved prognosis. These findings support the view that karyotypic diversity, necessary to adapt to selective pressures, may be limited in tumors so as to reduce the risk of excessive instability. This review aims to highlight the contribution of specialized DNA polymerases in limiting extreme genetic instability by allowing DNA replication to occur even in the presence of DNA damage, to either avoid broken forks or favor their repair after collapse. These mechanisms and their key regulators Rad18 and Polθ not only offer diversity and evolutionary advantage by increasing mutagenic events, but also provide cancer cells with a way to escape anti-cancer therapies that target replication forks. [ABSTRACT FROM AUTHOR]

Simple Summary: Recently, several molecules have improved the clinical outcome of acute myeloid leukemia (AML) patients. Despite these recent advances, their prognosis remains poor and new strategies to improve the standard anthracycline and Ara-C-based chemotherapy are needed. We recently published that dendrogenin A (DDA), a mammalian cholesterol metabolite with tumor-suppressor properties, can potentiate the effect of Ara-C to kill AML cells. In this study, we find that DDA can also potentiate anthracycline against AML. The potentiation of Ara-C by DDA is due to a switch from a protective autophagy to a deadly autophagy. Regarding anthracyclines, the potentiation of daunorubicin is caused by the modulation of the efflux by the PgP pump, and that of idarubicin, to an increase in DNA damage and to the induction of a rapid and lethal autophagy. This is caused by rapid modulation of AKT/mTOR and JNK activity, two major pathways involved both in DNA repair and lethal autophagy. Dendrogenin A (DDA), a mammalian cholesterol metabolite with tumor suppressor properties, has recently been shown to exhibit strong anti-leukemic activity in acute myeloid leukemia (AML) cells by triggering lethal autophagy. Here, we demonstrated that DDA synergistically enhanced the toxicity of anthracyclines in AML cells but not in normal hematopoietic cells. Combination index of DDA treatment with either daunorubicin or idarubicin indicated a strong synergism in KG1a, KG1 and MV4-11 cell lines. This was confirmed in vivo using immunodeficient mice engrafted with MOLM-14 cells as well as in a panel of 20 genetically diverse AML patient samples. This effect was dependent on Liver X Receptor β, a major target of DDA. Furthermore, DDA plus idarubicin strongly increased p53BP1 expression and the number of DNA strand breaks in alkaline comet assays as compared to idarubicin alone, whereas DDA alone was non-genotoxic. Mechanistically, DDA induced JNK phosphorylation and the inhibition of AKT phosphorylation, thereby maximizing DNA damage induced by idarubicin and decreasing DNA repair. This activated autophagic cell death machinery in AML cells. Overall, this study shows that the combination of DDA and idarubicin is highly promising and supports clinical trials of dendrogenin A in AML patients. [ABSTRACT FROM AUTHOR]

The effectiveness of checkpoint kinase 1 (Chk1) inhibitors at killing cancer cells is considered to be fully dependent on their effect on DNA replication initiation. Chk1 inhibition boosts origin firing, presumably limiting the availability of nucleotides and in turn provoking the slowdown and subsequent collapse of forks, thus decreasing cell viability. Here we show that slow fork progression in Chk1‐inhibited cells is not an indirect effect of excess new origin firing. Instead, fork slowdown results from the accumulation of replication barriers, whose bypass is impeded by CDK‐dependent phosphorylation of the specialized DNA polymerase eta (Polη). Also in contrast to the linear model, the accumulation of DNA damage in Chk1‐deficient cells depends on origin density but is largely independent of fork speed. Notwithstanding this, origin dysregulation contributes only mildly to the poor proliferation rates of Chk1‐depleted cells. Moreover, elimination of replication barriers by downregulation of helicase components, but not their bypass by Polη, improves cell survival. Our results thus shed light on the molecular basis of the sensitivity of tumors to Chk1 inhibition. Synopsis: Checkpoint Kinase 1 (Chk1) controls new origin firing and replication fork progression by independent mechanisms. In Chk1‐deficient cells, both DNA damage response defects and generation of replication barriers synergize to promote cell death. Excess chromatin association of replicative helicase cofactor CDC45 in Chk1‐deficient cells creates replication barriers.Replication roadblocks slow down fork elongation in Chk1‐deficient cells.Slow fork elongation in Chk1‐deficient cells is not causally related to surplus new origin firing.Replication barriers compromise survival of Chk1‐deficient cells. [ABSTRACT FROM AUTHOR]

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20–30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or "dormant" origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress. [ABSTRACT FROM AUTHOR]

Human DNA polymerase η (Polη) modulates susceptibility to skin cancer by promoting translesion DNA synthesis (TLS) past sunlight-induced cyclobutane pyrimidine dimers. Despite its well-established role in TLS synthesis, the role of Polη in maintaining genome stability in the absence of external DNA damage has not been well explored. We show here that short hairpin RNA-mediated depletion of Pol from undamaged human cells affects cell cycle progression and the rate of cell proliferation and results in increased spontaneous chromosome breaks and common fragile site expression with the activation of ATM-mediated DNA damage checkpoint signaling. These phenotypes were also observed in association with modified replication factory dynamics during S phase. In contrast to that seen in Polη-depleted cells, none of these cellular or karyotypic defects were observed in cells depleted for Pol℩, the closest relative of Polη. Our results identify a new role for Polη in maintaining genomic stability during unperturbed S phase and challenge the idea that the sole functional role of Polη in human cells is in TLS DNA damage tolerance and/or repair pathways following exogenous DNA damage. [ABSTRACT FROM AUTHOR]

The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21's PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis. [ABSTRACT FROM AUTHOR]

Human DNA polymerase η (Pol η) is best known for its role in responding to UV irradiation-induced genome damage. We have recently observed that Pol η is also required for the stability of common fragile sites (CFSs), whose rearrangements are considered a driving force of oncogenesis. Here, we explored the molecular mechanisms underlying this newly identified role. We demonstrated that Pol η accumulated at CFSs upon partial replication stress and could efficiently replicate non-B DNA sequences within CFSs. Pol η deficiency led to persistence of checkpoint-blind under-replicated CFS regions in mitosis, detectable as FANCD2-associated chromosomal sites that were transmitted to daughter cells in 53BP1-shielded nuclear bodies. Expression of a catalytically inactive mutant of Pol η increased replication fork stalling and activated the replication checkpoint. These data are consistent with the requirement of Pol η-dependent DNA synthesis during S phase at replication forks stalled in CFS regions to suppress CFS instability by preventing checkpoint-blind under-replicated DNA in mitosis. [ABSTRACT FROM AUTHOR]

Abstract: Specific DNA repair pathways from Trypanosoma cruzi are believed to protect genomic DNA and kinetoplast DNA (kDNA) from mutations. Particular pathways are supposed to operate in order to repair nucleotides oxidized by reactive oxygen species (ROS) during parasite infection, being 7,8-dihydro-8-oxoguanine (8oxoG) a frequent and highly mutagenic base alteration. If unrepaired, 8oxoG can lead to cytotoxic base transversions during DNA replication. In mammals, DNA polymerase beta (Polβ) is mainly involved in base excision repair (BER) of oxidative damage. However its biological role in T. cruzi is still unknown. We show, by immunofluorescence localization, that T. cruzi DNA polymerase beta (Tcpolβ) is restricted to the antipodal sites of kDNA in replicative epimastigote and amastigote developmental stages, being strictly localized to kDNA antipodal sites between G1/S and early G2 phase in replicative epimastigotes. Nevertheless, this polymerase was detected inside the mitochondrial matrix of trypomastigote forms, which are not able to replicate in culture. Parasites over expressing Tcpolβ showed reduced levels of 8oxoG in kDNA and an increased survival after treatment with hydrogen peroxide when compared to control cells. However, this resistance was lost after treating Tcpolβ overexpressors with methoxiamine, a potent BER inhibitor. Curiously, a presumed DNA repair focus containing Tcpolβ was identified in the vicinity of kDNA of cultured wild type epimastigotes after treatment with hydrogen peroxide. Taken together our data suggest participation of Tcpolβ during kDNA replication and repair of oxidative DNA damage induced by genotoxic stress in this organelle. [Copyright &y& Elsevier]