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Nucleic Acids Research Jul 2018Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and...
Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj-/- cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj-/- cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.
Topics: Adenosine Triphosphatases; Animals; Aphidicolin; Cell Line; Checkpoint Kinase 1; Chickens; Cisplatin; DNA Breaks, Double-Stranded; DNA Helicases; DNA Repair; DNA Replication; DNA, Single-Stranded; Fanconi Anemia; Fanconi Anemia Complementation Group Proteins; G-Quadruplexes; Mutation, Missense; Oxazoles; RNA Helicases; Rad51 Recombinase; Recombinases; Replication Protein A; Stress, Physiological
PubMed: 29788478
DOI: 10.1093/nar/gky403 -
BMC Genomics Jul 2019Replication stress (RS) gives rise to DNA damage that threatens genome stability. RS can originate from different sources that stall replication by diverse mechanisms....
BACKGROUND
Replication stress (RS) gives rise to DNA damage that threatens genome stability. RS can originate from different sources that stall replication by diverse mechanisms. However, the mechanism underlying how different types of RS contribute to genome instability is unclear, in part due to the poor understanding of the distribution and characteristics of damage sites induced by different RS mechanisms.
RESULTS
We use ChIP-seq to map γH2AX binding sites genome-wide caused by aphidicolin (APH), hydroxyurea (HU), and methyl methanesulfonate (MMS) treatments in human lymphocyte cells. Mapping of γH2AX ChIP-seq reveals that APH, HU, and MMS treatments induce non-random γH2AX chromatin binding at discrete regions, suggesting that there are γH2AX binding hotspots in the genome. Characterization of the distribution and sequence/epigenetic features of γH2AX binding sites reveals that the three treatments induce γH2AX binding at largely non-overlapping regions, suggesting that RS may cause damage at specific genomic loci in a manner dependent on the fork stalling mechanism. Nonetheless, γH2AX binding sites induced by the three treatments share common features including compact chromatin, coinciding with larger-than-average genes, and depletion of CpG islands and transcription start sites. Moreover, we observe significant enrichment of SINEs in γH2AX sites in all treatments, indicating that SINEs may be a common barrier for replication polymerases.
CONCLUSIONS
Our results identify the location and common features of genome instability hotspots induced by different types of RS, and help in deciphering the mechanisms underlying RS-induced genetic diseases and carcinogenesis.
Topics: Aphidicolin; Binding Sites; Cell Line; Chromosome Mapping; DNA Replication; Genome, Human; Genomic Instability; Histones; Humans; Hydroxyurea; Stress, Physiological; Sulfinic Acids
PubMed: 31299901
DOI: 10.1186/s12864-019-5934-4 -
Plant Physiology Nov 2011The CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene of Arabidopsis (Arabidopsis thaliana) encodes a putative membrane protein of unknown biochemical...
The CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene of Arabidopsis (Arabidopsis thaliana) encodes a putative membrane protein of unknown biochemical function and displays highly pleiotropic functions, particularly in pathogen responses, cell proliferation, cell expansion, and cell death. Here, we demonstrate a link between CPR5 and the GLABRA1 ENHANCER BINDING PROTEIN (GeBP) family of transcription factors. We investigated the primary role of the GeBP/GeBP-like (GPL) genes using transcriptomic analysis of the quadruple gebp gpl1,2,3 mutant and one overexpressing line that displays several cpr5-like phenotypes including dwarfism, spontaneous necrotic lesions, and increased pathogen resistance. We found that GeBP/GPLs regulate a set of genes that represents a subset of the CPR5 pathway. This subset includes genes involved in response to stress as well as cell wall metabolism. Analysis of the quintuple gebp gpl1,2,3 cpr5 mutant indicates that GeBP/GPLs are involved in the control of cell expansion in a CPR5-dependent manner but not in the control of cell proliferation. In addition, to our knowledge, we provide the first evidence that the CPR5 protein is localized in the nucleus of plant cells and that a truncated version of the protein with no transmembrane domain can trigger cpr5-like processes when fused to the VP16 constitutive transcriptional activation domain. Our results provide clues on how CPR5 and GeBP/GPLs play opposite roles in the control of cell expansion and suggest that the CPR5 protein is involved in transcription.
Topics: Aphidicolin; Arabidopsis; Arabidopsis Proteins; Cell Size; DNA-Binding Proteins; Epistasis, Genetic; Gene Expression Regulation, Plant; Genes, Plant; Membrane Proteins; Mutation; Nuclear Proteins; Phenotype; Plant Epidermis; Protein Transport; Signal Transduction; Transcription Factors; Transcription, Genetic
PubMed: 21875893
DOI: 10.1104/pp.111.179804 -
Eukaryotic Cell Apr 2008Giardia intestinalis is a ubiquitous intestinal protozoan parasite and has been proposed to represent the earliest diverging lineage of extant eukaryotes. Despite the...
Giardia intestinalis is a ubiquitous intestinal protozoan parasite and has been proposed to represent the earliest diverging lineage of extant eukaryotes. Despite the importance of Giardia as a model organism, research on Giardia has been hampered by an inability to achieve cell cycle synchrony for in vitro cultures. This report details successful methods for attaining cell cycle synchrony in Giardia cultures. The research presented here demonstrates reversible cell cycle arrest in G(1)/S and G(2)/M with aphidicolin and nocodazole, respectively. Following synchronization, cells were able to recover completely from drug treatment and remained viable and maintained synchronous growth for 6 h. These techniques were used to synchronize Giardia cultures to increase the percentages of mitotic spindles in the cultures. This method of synchronization will enhance our ability to study cell cycle-dependent processes in G. intestinalis.
Topics: Animals; Aphidicolin; Cell Cycle; Cell Survival; Enzyme Inhibitors; Flow Cytometry; Giardia lamblia; Nocodazole; Tubulin Modulators
PubMed: 18296622
DOI: 10.1128/EC.00415-07 -
The Journal of Biological Chemistry Aug 2018In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks...
In growing cells, DNA replication precedes mitotic cell division to transmit genetic information to the next generation. The slowing or stalling of DNA replication forks at natural or exogenous obstacles causes "replicative stress" that promotes genomic instability and affects cellular fitness. Replicative stress phenotypes can be characterized at the single-molecule level with DNA combing or stretched DNA fibers, but interpreting the results obtained with these approaches is complicated by the fact that the speed of replication forks is connected to the frequency of origin activation. Primary alterations in fork speed trigger secondary responses in origins, and, conversely, primary alterations in the number of active origins induce compensatory changes in fork speed. Here, by employing interventions that temporally restrict either fork speed or origin firing while still allowing interrogation of the other variable, we report a set of experimental conditions to separate cause and effect in any manipulation that affects DNA replication dynamics. Using HeLa cells and chemical inhibition of origin activity (through a CDC7 kinase inhibitor) and of DNA synthesis (via the DNA polymerase inhibitor aphidicolin), we found that primary effects of replicative stress on velocity of replisomes (fork rate) can be readily distinguished from primary effects on origin firing. Identifying the primary cause of replicative stress in each case as demonstrated here may facilitate the design of methods to counteract replication stress in primary cells or to enhance it in cancer cells to increase their susceptibility to therapies that target DNA repair.
Topics: Aphidicolin; Cell Cycle Proteins; Cellular Senescence; DNA; DNA Repair; DNA Replication; HeLa Cells; Humans; Protein Serine-Threonine Kinases; Replication Origin
PubMed: 29959228
DOI: 10.1074/jbc.RA118.003740 -
The Plant Cell Jan 2002All living organisms have to protect the integrity of their genomes from a wide range of genotoxic stresses to which they are inevitably exposed. However, understanding... (Comparative Study)
Comparative Study
All living organisms have to protect the integrity of their genomes from a wide range of genotoxic stresses to which they are inevitably exposed. However, understanding of DNA repair in plants lags far behind such knowledge in bacteria, yeast, and mammals, partially as a result of the absence of efficient in vitro systems. Here, we report the experimental setup for an Arabidopsis in vitro repair synthesis assay. The repair of plasmid DNA treated with three different DNA-damaging agents, UV light, cisplatin, and methylene blue, after incubation with whole-cell extract was monitored. To validate the reliability of our assay, we analyzed the repair proficiency of plants depleted in AtRAD1 activity. The reduced repair of UV light- and cisplatin-damaged DNA confirmed the deficiency of these plants in nucleotide excision repair. Decreased repair of methylene blue-induced oxidative lesions, which are believed to be processed by the base excision repair machinery in mammalian cells, may indicate a possible involvement of AtRAD1 in the repair of oxidative damage. Differences in sensitivity to DNA polymerase inhibitors (aphidicolin and dideoxy TTP) between plant and human cell extracts were observed with this assay.
Topics: Aphidicolin; Arabidopsis; Cell Extracts; Cisplatin; DNA Damage; DNA Repair; DNA-Binding Proteins; Dideoxynucleotides; Endonucleases; Genome, Plant; Humans; Methylene Blue; Nucleic Acid Synthesis Inhibitors; Oxidative Stress; Plants, Genetically Modified; Thymine Nucleotides; Ultraviolet Rays
PubMed: 11826311
DOI: 10.1105/tpc.010258 -
BMC Cell Biology Apr 2010Cytometric measurements of DNA content and chromatin-bound Mcm2 have demonstrated bimodal patterns of expression in G1. These patterns, the replication licensing...
BACKGROUND
Cytometric measurements of DNA content and chromatin-bound Mcm2 have demonstrated bimodal patterns of expression in G1. These patterns, the replication licensing function of Mcm proteins, and a correlation between Mcm loading and cell cycle commitment for cells re-entering the cell cycle, led us to test the idea that cells expressing a defined high level of chromatin-bound Mcm6 in G1 are committed--i.e., past the G1 restriction point. We developed a cell-based assay for tightly-bound PCNA (PCNA*) and Mcm6 (Mcm6*), DNA content, and a mitotic marker to clearly define G1, S, G2, and M phases of the cell cycle. hTERT-BJ1, hTERT-RPE-1, and Molt4 cells were extracted with Triton X-100 followed by methanol fixation, stained with antibodies and DAPI, then measured by cytometry.
RESULTS
Bivariate analysis of cytometric data demonstrated complex patterns with distinct clustering for all combinations of the 4 variables. In G1, cells clustered in two groups characterized by low and high Mcm6* expression. Serum starvation and release experiments showed that residence in the high group was in late G1, just prior to S phase. Kinetic experiments, employing serum withdrawal, and stathmokinetic analysis with aphidicolin, mimosine or nocodazole demonstrated that cells with high levels of Mcm6* cycled with the committed phases of the cell cycle (S, G2, and M).
CONCLUSIONS
A multivariate assay for Mcm6*, PCNA*, DNA content, and a mitotic marker provides analysis capable of estimating the fraction of pre and post-restriction point G1 cells and supports the idea that there are at least two states in G1 defined by levels of chromatin bound Mcm proteins.
Topics: Antineoplastic Agents; Aphidicolin; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Separation; Chromatin; Culture Media, Serum-Free; DNA; Flow Cytometry; G1 Phase; Humans; Lymphoma, T-Cell; Mimosine; Minichromosome Maintenance Complex Component 6; Nocodazole; Proliferating Cell Nuclear Antigen; Protein Binding
PubMed: 20398392
DOI: 10.1186/1471-2121-11-26 -
Nucleic Acids Research Apr 2019Werner syndrome (WS) is a cancer-prone disease caused by deficiency of Werner protein (WRN). WRN maintains genome integrity by promoting replication-fork stability after...
Werner syndrome (WS) is a cancer-prone disease caused by deficiency of Werner protein (WRN). WRN maintains genome integrity by promoting replication-fork stability after various forms of replication stress. Under mild replication stress, WS cells show impaired ATR-mediated CHK1 activation. However, it remains unclear if WS cells elicit other repair pathway. We demonstrate that loss of WRN leads to enhanced ATM phosphorylation upon prolonged exposure to aphidicolin, a specific inhibitor of DNA polymerases, resulting in CHK1 activation. Moreover, we find that loss of WRN sensitises cells to replication-transcription collisions and promotes accumulation of R-loops, which undergo XPG-dependent cleavage responsible for ATM signalling activation. Importantly, we observe that ATM pathway limits chromosomal instability in WS cells. Finally, we prove that, in WS cells, genomic instability enhanced upon chemical inhibition of ATM kinase activity is counteracted by direct or indirect suppression of R-loop formation or by XPG abrogation. Together, these findings suggest a potential role of WRN as regulator of R-loop-associated genomic instability, strengthening the notion that conflicts between replication and transcription can affect DNA replication, leading to human disease and cancer.
Topics: Aphidicolin; Ataxia Telangiectasia Mutated Proteins; Checkpoint Kinase 1; DNA Damage; DNA Replication; Fibroblasts; Gene Expression Regulation; Genomic Instability; Humans; Phosphorylation; Signal Transduction; Werner Syndrome; Werner Syndrome Helicase
PubMed: 30657978
DOI: 10.1093/nar/gkz025 -
Molecular and Cellular Biology Aug 2012RMI1 is a member of an evolutionarily conserved complex composed of BLM and topoisomerase IIIα (TopoIIIα). This complex exhibits strand passage activity in vitro,...
RMI1 is a member of an evolutionarily conserved complex composed of BLM and topoisomerase IIIα (TopoIIIα). This complex exhibits strand passage activity in vitro, which is likely important for DNA repair and DNA replication in vivo. The inactivation of RMI1 causes genome instability, including elevated levels of sister chromatid exchange and accelerated tumorigenesis. Using molecular combing to analyze DNA replication at the single-molecule level, we show that RMI1 is required to promote normal replication fork progression. The fork progression defect in RMI1-depleted cells is alleviated in cells lacking BLM, indicating that RMI1 functions downstream of BLM in promoting replication elongation. RMI1 localizes to subnuclear foci with BLM and TopoIIIα in response to replication stress. The proper localization of the complex requires a BLM-TopoIIIα-RMI1 interaction and is essential for RMI1 to promote recovery from replication stress. These findings reveal direct roles of RMI1 in DNA replication and the replication stress response, which could explain the molecular basis for its involvement in suppressing sister chromatid exchange and tumorigenesis.
Topics: Aphidicolin; Carrier Proteins; Cell Line; DNA Replication; DNA Topoisomerases, Type I; DNA-Binding Proteins; HEK293 Cells; Humans; Nuclear Proteins; RNA Interference; RNA, Small Interfering; RecQ Helicases; Sister Chromatid Exchange; Stress, Mechanical
PubMed: 22645306
DOI: 10.1128/MCB.00255-12 -
PloS One 2012Our previous study demonstrated that 45S ribosomal DNA (45S rDNA) clusters were chromosome fragile sites expressed spontaneously in Lolium. In this study, fragile...
Our previous study demonstrated that 45S ribosomal DNA (45S rDNA) clusters were chromosome fragile sites expressed spontaneously in Lolium. In this study, fragile phenotypes of 45S rDNA were observed under aphidicolin (APH) incubation in several plant species. Further actinomycin D (ActD) treatment showed that transcriptional stress might interfere with chromatin packaging, resulting in 45S rDNA fragile expression. These data identified 45S rDNA sites as replication-dependent as well as transcription-dependent fragile sites in plants. In the presence of ActD, a dramatic switch to an open chromatin conformation and accumulated incomplete 5' end of the external transcribed spacer (5'ETS) transcripts were observed, accompanied by decreased DNA methylation, decreased levels of histone H3, and increased histone acetylation and levels of H3K4me2, suggesting that these epigenetic alterations are associated with failure of 45S rDNA condensation. Furthermore, the finding that γ-H2AX was accumulated at 45S rDNA sites following ActD treatment suggested that the DNA damage signaling pathway was associated with the appearance of 45S rDNA fragile phenotypes. Our data provide a link between 45S rDNA transcription and chromatin-packaging defects and open the door for further identifying the molecular mechanism involved.
Topics: Aphidicolin; Chromatin; Chromosome Fragile Sites; DNA Methylation; Dactinomycin; Epigenesis, Genetic; Gene Expression Regulation, Plant; Genome, Plant; Genomic Instability; Histone-Lysine N-Methyltransferase; Histones; Lolium; RNA, Ribosomal
PubMed: 22509394
DOI: 10.1371/journal.pone.0035139