scholarly journals DNMT3A with Leukemia-Associated Mutations Directs Sensitivity to DNA Damage at Replication Forks

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 535-535
Author(s):  
Kartika Venugopal ◽  
Daniil E Shabashvili ◽  
Jianping Li ◽  
Luisa M Posada ◽  
Richard Lynn Bennett ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
Replication Stress ◽  
S Phase ◽  
Differential Sensitivity ◽  
Mutant Cells ◽  
In Cells

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novoacute myeloid leukemia (AML) and are associated with poor prognosis. Although studies demonstrated survival benefit of induction chemotherapy dose intensification, outcomes remain unsatisfactory in most patients due to advanced age, comorbidities, and hence inability to tolerate treatment. Clinical trials of low-intensity regimens combining cytarabine and cladribine, nucleoside analog chain terminators that stall DNA replication, appear to be safe and effective, and tend to particularly benefit patients with DNMT3Amutations. Consistently, we observe increased sensitivity to cytarabine, fludarabine, and cladribine in multiple cellular systems harboring mutant DNMT3Ain vitro (Figure 1A, B). Differential sensitivity to cytarabine was confirmed in normal and leukemic primary bone marrow cells derived from mice with and without Dnmt3a mutations ex vivo (Figure 1C). Dynamic chromatin organization plays a pivotal role in DNA-associated cellular processes including DNA replication and damage repair. We previously found altered chromatin remodeling in cells expressing mutant DNMT3A after genotoxic stress. Gene expression studies by us and others demonstrated negative enrichment of cell cycle related signatures including G2/M checkpoint adaptation, in cells with DNMT3A mutations. These signatures are implicated in DNA damage response and replication fork integrity and suggest sensitivity to replication stress. To investigate the mechanism of differential sensitivity to cytarabine-induced DNA damage, we overexpressed wildtype (WT) or R882 mutant (MUT) forms of DNMT3A in U2OS cells, a well-established model for DNA damage studies. Analysis of the DNA damage signaling proficiency in response to cytarabine revealed persistent intra-S phase checkpoint activation (phospho-CHK1), accompanied by accumulation of DNA damage visualized by γH2A.X foci and by Comet assay in the DNMT3A(mut) overexpression cells (Figure 1D). This damage was only partially resolved after drug had been removed and cells were allowed to repair the DNA (Figure 1E), and was carried through mitosis, resulting in increased rate of micronucleation.At the same time, DNMT3A mutant cells remained proficient in initiating homology-directed repair (HDR) and non-homologous end joining (NHEJ) pathways, evidenced by RAD51 and 53BP-1 foci formation, respectively. These data demonstrate enhanced sensitivity to cytarabine in cells expressing mutant DNMT3A is due to increased susceptibility to DNA damage during replication, rather than defects in double-strand DNA break repair. In support of this, cells with mutant DNMT3Awere characterized by accentuated replication stress as evidenced by high levels of phospho-RPA, which persisted after drug wash-out (Figure 1F). Consistently, DNMT3A-mutant cells treated with cytarabine were characterized by a higher number and a larger area of PCNA foci. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that while the overall kinetics of replication restart remained unchanged, cells with DNMT3A(mut) showed higher rate of fork collapse and increased reliance on latent replication origins (Figure 1G). Gene expression profiling by RNA-seq identified dysregulation of pathways associated with cell cycle progression, specifically G1/S phase transition, DNA replication, DNA integrity checkpoint, and chromatin. Our studies show cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication and suggest that cytarabine-induced replication fork stalling may further synergize with other agents aimed at DNA damage and replication. Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals DNMT3A harboring leukemia-associated mutations directs sensitivity to DNA damage at replication forks

2021 ◽  
Author(s):  
Kartika Venugopal ◽  
Pawel Nowialis ◽  
Yang Feng ◽  
Daniil E Shabashvili ◽  
Cassandra M Berntsen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Dna Damage ◽  
Replication Fork ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Nucleoside Analogs ◽  
Cellular Systems ◽  
Double Labeling ◽  
In Cells

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

scholarly journals Regulation of DNA Replication Licensing and Re-Replication by Cdt1

2021 ◽  
Vol 22 (10) ◽  
pp. 5195
Author(s):  
Hui Zhang
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
E3 Ligase ◽  
S Phase ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Repair Synthesis ◽  
Ubiquitin E3 Ligase

In eukaryotic cells, DNA replication licensing is precisely regulated to ensure that the initiation of genomic DNA replication in S phase occurs once and only once for each mitotic cell division. A key regulatory mechanism by which DNA re-replication is suppressed is the S phase-dependent proteolysis of Cdt1, an essential replication protein for licensing DNA replication origins by loading the Mcm2-7 replication helicase for DNA duplication in S phase. Cdt1 degradation is mediated by CRL4Cdt2 ubiquitin E3 ligase, which further requires Cdt1 binding to proliferating cell nuclear antigen (PCNA) through a PIP box domain in Cdt1 during DNA synthesis. Recent studies found that Cdt2, the specific subunit of CRL4Cdt2 ubiquitin E3 ligase that targets Cdt1 for degradation, also contains an evolutionarily conserved PIP box-like domain that mediates the interaction with PCNA. These findings suggest that the initiation and elongation of DNA replication or DNA damage-induced repair synthesis provide a novel mechanism by which Cdt1 and CRL4Cdt2 are both recruited onto the trimeric PCNA clamp encircling the replicating DNA strands to promote the interaction between Cdt1 and CRL4Cdt2. The proximity of PCNA-bound Cdt1 to CRL4Cdt2 facilitates the destruction of Cdt1 in response to DNA damage or after DNA replication initiation to prevent DNA re-replication in the cell cycle. CRL4Cdt2 ubiquitin E3 ligase may also regulate the degradation of other PIP box-containing proteins, such as CDK inhibitor p21 and histone methylase Set8, to regulate DNA replication licensing, cell cycle progression, DNA repair, and genome stability by directly interacting with PCNA during DNA replication and repair synthesis.

scholarly journals Set1-dependent H3K4 methylation becomes critical for DNA replication fork progression in response to changes in S phase dynamics in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Christophe de La Roche Saint-André ◽  
Vincent Géli
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
Genetic Material ◽  
S Phase ◽  
Replication Forks ◽  
H3k4 Methylation ◽  
Origin Firing ◽  
Phase Dynamics

AbstractDNA replication is a highly regulated process that occurs in the context of chromatin structure and is sensitive to several histone post-translational modifications. In Saccharomyces cerevisiae, the histone methylase Set1 is responsible for the transcription-dependent deposition of H3K4 methylation (H3K4me) throughout the genome. Here we show that a combination of a hypomorphic replication mutation (orc5-1) with the absence of Set1 (set1Δ) compromises the progression through S phase, and this is associated with a large increase in DNA damage. The ensuing DNA damage checkpoint activation, in addition to that of the spindle assembly checkpoint, restricts the growth of orc5-1 set1Δ. Interestingly, orc5-1 set1Δ is sensitive to the lack of RNase H activity while a reduction of histone levels is able to counterbalance the loss of Set1. We propose that the recently described Set1-dependent mitigation of transcription-replication conflicts becomes critical for growth when the replication forks accelerate due to decreased origin firing in the orc5-1 background. Furthermore, we show that an increase of reactive oxygen species (ROS) levels, likely a consequence of the elevated DNA damage, is partly responsible for the lethality in orc5-1 set1Δ.Author summaryDNA replication, that ensures the duplication of the genetic material, starts at discrete sites, termed origins, before proceeding at replication forks whose progression is carefully controlled in order to avoid conflicts with the transcription of genes. In eukaryotes, DNA replication occurs in the context of chromatin, a structure in which DNA is wrapped around proteins, called histones, that are subjected to various chemical modifications. Among them, the methylation of the lysine 4 of histone H3 (H3K4) is carried out by Set1 in Saccharomyces cerevisiae, specifically at transcribed genes. We report that, when the replication fork accelerates in response to a reduction of active origins, the absence of Set1 leads to accumulation of DNA damage. Because H3K4 methylation was recently shown to slow down replication at transcribed genes, we propose that the Set1-dependent becomes crucial to limit the occurrence of conflicts between replication and transcription caused by replication fork acceleration. In agreement with this model, stabilization of transcription-dependent structures or reduction histone levels, to limit replication fork velocity, respectively exacerbates or moderates the effect of Set1 loss. Last, but not least, we show that the oxidative stress associated to DNA damage is partly responsible for cell lethality.

Reducing MCM Loading Causes Chromosomal Aneuploidy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3349-3349
Author(s):  
Stephen J. Orr ◽  
Terry Gaymes ◽  
Rong Wang ◽  
Barbara Czepulkowski ◽  
Darius Ladon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
T Cells ◽  
Dna Replication ◽  
Genomic Instability ◽  
Chromosomal Abnormalities ◽  
S Phase ◽  
Human T Cells ◽  
Dna Damage Responses ◽  
Mcm Proteins

Abstract Normal DNA replication must be accurate and occur only once per cell cycle. Sites of DNA replication are specified by binding the origin recognition complex, that includes minichromosome maintenance (MCM) proteins. Paradoxically, in higher eukaryotes MCM proteins are present in >20 fold excess of that required for DNA replication. They are also downregulated by elevated expression of proteins such as cyclin E that occurs in cancers, including AML and breast cancer. We investigated why human cells need “excess” MCM proteins and whether the reduction of MCM protein levels might contribute to a malignant phenotype. We determined the consequences of reducing the levels of MCM proteins in primary human T cells in which cell cycle controls and DNA damage responses are normal. Mass spectrometry sequencing of chromatin/nuclear matrix-bound proteins and western blotting identified that Mcm7 is not present in quiescent, normal primary human T cells. Mcm7 is induced in mid G1after the G0→G1 commitment point, the point beyond which T cells are committed to entering the cell cycle. Reduction of Mcm7 with siRNA to <5% of normal during G0→G1→S-phase reduces chromatin-binding of each of the MCM proteins that form the DNA helicase. However, these cells still enter S-phase and replicate DNA. Reducing MCM levels by titrating siRNA causes dose-dependent DNA-damage responses involving activation of ATR & ATM and Chk1 & Chk2. However, cells depleted of Mcm7 do not undergo apoptosis, rather reducing MCM levels even by 50% causes gross non-clonal chromosomal abnormalities normally found in genomic instability syndromes. M-FISH identified chromosome translocations, as well as loss and gain of individual chromosomes, which can occur individually or together in the same cell. Reducing MCM levels also causes misrepair by non-homologous end joining (NHEJ), and both NHEJ and homologous recombination (HR) are necessary for chromosomal abnormalities to occur. Therefore, “excess” MCM proteins that are present in a normal, proliferating cell are necessary for maintaining genome stability and reduction of MCM loading onto DNA that occurs in cancers is sufficient to cause genomic instability.

scholarly journals Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks

2016 ◽  
Vol 113 (26) ◽  
pp. E3676-E3685 ◽  
Author(s):  
Nicholas A. Willis ◽  
Chunshui Zhou ◽  
Andrew E. H. Elia ◽  
Johanne M. Murray ◽  
Antony M. Carr ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Dna Replication ◽  
Fission Yeast ◽  
Dna Damage Response ◽  
Cellular Response ◽  
Genome Stability ◽  
Biological Significance ◽  
S Phase ◽  
Damage Response

The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase–specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes.

scholarly journals Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

2008 ◽  
Vol 82 (18) ◽  
pp. 9056-9064 ◽  
Author(s):  
Sally Roberts ◽  
Sarah R. Kingsbury ◽  
Kai Stoeber ◽  
Gillian L. Knight ◽  
Phillip H. Gallimore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Host Cell ◽  
S Phase ◽  
Human Papillomaviruses ◽  
Soluble Form ◽  
Cellular Dna ◽  
In Cells

ABSTRACT Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

scholarly journals A Mutant Allele of MRE11 Found in Mismatch Repair-deficient Tumor Cells Suppresses the Cellular Response to DNA Replication Fork Stress in a Dominant Negative Manner

2008 ◽  
Vol 19 (4) ◽  
pp. 1693-1705 ◽  
Author(s):  
Qin Wen ◽  
Jennifer Scorah ◽  
Geraldine Phear ◽  
Gary Rodgers ◽  
Sheila Rodgers ◽  
...  
Keyword(s):  
Dna Replication ◽  
Mutant Allele ◽  
Cellular Response ◽  
Replication Fork ◽  
Protein A ◽  
Cancer Cell Line ◽  
Replication Stress ◽  
Dominant Negative ◽  
Mutant Protein ◽  
In Cells

The interaction of ataxia-telangiectasia mutated (ATM) and the Mre11/Rad50/Nbs1 (MRN) complex is critical for the response of cells to DNA double-strand breaks; however, little is known of the role of these proteins in response to DNA replication stress. Here, we report a mutant allele of MRE11 found in a colon cancer cell line that sensitizes cells to agents causing replication fork stress. The mutant Mre11 weakly interacts with Rad50 relative to wild type and shows little affinity for Nbs1. The mutant protein lacks 3′-5′ exonuclease activity as a result of loss of part of the conserved nuclease domain; however, it retains binding affinity for single-stranded DNA (ssDNA), double-stranded DNA with a 3′ single-strand overhang, and fork-like structures containing ssDNA regions. In cells, the mutant protein shows a time- and dose-dependent accumulation in chromatin after thymidine treatment that corresponds with increased recruitment and hyperphosphorylation of replication protein A. ATM autophosphorylation, Mre11 foci, and thymidine-induced homologous recombination are suppressed in cells expressing the mutant allele. Together, our results suggest that the mutant Mre11 suppresses the cellular response to replication stress by binding to ssDNA regions at disrupted forks and impeding replication restart in a dominant negative manner.

scholarly journals Activation of the DNA Damage Checkpoint in Mutants Defective in DNA Replication Initiation

2008 ◽  
Vol 19 (10) ◽  
pp. 4374-4382 ◽  
Author(s):  
Ling Yin ◽  
Alexandra Monica Locovei ◽  
Gennaro D'Urso
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Replication Fork ◽  
S Phase ◽  
Replication Initiation ◽  
Dna Damage Checkpoint ◽  
Origin Firing ◽  
Dna Replication Initiation ◽  
Fork Stalling ◽  
S Phase Checkpoint

In the fission yeast, Schizosaccharomyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both prevent premature entry into mitosis and to stabilize paused replication forks. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, mutants defective in DNA replication initiation require the Chk1 kinase. This suggests that defects in DNA replication initiation can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. We refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). Chk1 is active in rid mutants, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, a protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation.

scholarly journals Protein phosphatase 2A (PP2A) promotes anaphase entry after DNA replication stress in budding yeast

2021 ◽  
Author(s):  
Cory Haluska ◽  
Fengzhi Jin ◽  
Yanchang Wang
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Protein Phosphatase ◽  
Budding Yeast ◽  
Replication Stress ◽  
S Phase ◽  
Viability Loss ◽  
Phosphatase 2A ◽  
Dna Replication Stress

DNA replication stress activates the S-phase checkpoint that arrests the cell cycle, but it is poorly understood how cells recover from this arrest. Cyclin-dependent kinase (CDK) and Protein Phosphatase 2A (PP2A) are key cell cycle regulators, and Cdc55 is a regulatory subunit of PP2A in budding yeast. We found that yeast cells lacking functional PP2ACdc55 showed slow growth in the presence of hydroxyurea (HU), a DNA synthesis inhibitor, without obvious viability loss. Moreover, PP2A mutants exhibited delayed anaphase entry and sustained levels of anaphase inhibitor Pds1 after HU treatment. A DNA damage checkpoint Chk1 phosphorylates and stabilizes Pds1. We showed that chk1Δ and mutation of the Chk1 phosphorylation sites in Pds1 largely restored efficient anaphase entry in PP2A mutants after HU treatment. In addition, deletion of SWE1 that encodes the inhibitory kinase for CDK or mutation of the Swe1 phosphorylation site in CDK ( cdc28F19) also suppressed the anaphase entry delay in PP2A mutants after HU treatment. Our genetic data suggest that Swe1/CDK acts upstream of Pds1. Surprisingly, cdc55Δ showed significant suppression to the viability loss of S-phase checkpoint mutants during DNA synthesis block. Together, our results uncover a PP2A-Swe1-CDK-Chk1-Pds1 axis that promotes recovery from DNA replication stress.

scholarly journals Replication fork dynamics and the DNA damage response

2012 ◽  
Vol 443 (1) ◽  
pp. 13-26 ◽  
Author(s):  
Rebecca M. Jones ◽  
Eva Petermann
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Replication Fork ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Replication Initiation ◽  
Developmental Defects ◽  
Replication Fork Progression ◽  
Damage Response

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.

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