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The Korean Journal of Parasitology Apr 2019To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and...
To identify the component(s) involved in cell cycle control in the protozoan Giardia lamblia, cells arrested at the G1/S- or G2-phase by treatment with nocodazole and aphidicolin were prepared from the synchronized cell cultures. RNA-sequencing analysis of the 2 stages of Giardia cell cycle identified several cell cycle genes that were up-regulated at the G2-phase. Transcriptome analysis of cells in 2 distinct cell cycle stages of G. lamblia confirmed previously reported components of cell cycle (PcnA, cyclin B, and CDK) and identified additional cell cycle components (NEKs, Mad2, spindle pole protein, and CDC14A). This result indicates that the cell cycle machinery operates in this protozoan, one of the earliest diverging eukaryotic lineages.
Topics: Antiprotozoal Agents; Aphidicolin; Cell Cycle Checkpoints; Cell Cycle Proteins; G2 Phase; Gene Expression Profiling; Giardia lamblia; Nocodazole; Sequence Analysis, RNA; Up-Regulation
PubMed: 31104412
DOI: 10.3347/kjp.2019.57.2.185 -
Cell Dec 2002Conditions that partially inhibit DNA replication induce expression of common fragile sites. These sites form gaps and breaks on metaphase chromosomes and are deleted...
Conditions that partially inhibit DNA replication induce expression of common fragile sites. These sites form gaps and breaks on metaphase chromosomes and are deleted and rearranged in many tumors. Yet, the mechanism of fragile site expression has been elusive. We demonstrate that the replication checkpoint kinase ATR, but not ATM, is critical for maintenance of fragile site stability. ATR deficiency results in fragile site expression with and without addition of replication inhibitors. Thus, we propose that fragile sites are unreplicated chromosomal regions resulting from stalled forks that escape the ATR replication checkpoint. These findings have important implications for understanding both the mechanism of fragile site instability and the consequences of stalled replication in mammalian cells.
Topics: 2-Aminopurine; Antimetabolites; Aphidicolin; Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Caffeine; Cell Cycle Proteins; Cells, Cultured; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes; DNA; DNA-Binding Proteins; Dose-Response Relationship, Drug; HeLa Cells; Humans; Metaphase; Microscopy, Fluorescence; Models, Biological; Phosphodiesterase Inhibitors; Protein Serine-Threonine Kinases; Tumor Suppressor Proteins
PubMed: 12526805
DOI: 10.1016/s0092-8674(02)01113-3 -
Nucleic Acids Research Jan 2021The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic...
The human genome contains hundreds of large, structurally diverse blocks that are insufficiently represented in the reference genome and are thus not amenable to genomic analyses. Structural diversity in the human population suggests that these blocks are unstable in the germline; however, whether or not these blocks are also unstable in the cancer genome remains elusive. Here we report that the 500 kb block called KRTAP_region_1 (KRTAP-1) on 17q12-21 recurrently demarcates the amplicon of the ERBB2 (HER2) oncogene in breast tumors. KRTAP-1 carries numerous tandemly-duplicated segments that exhibit diversity within the human population. We evaluated the fragility of the block by cytogenetically measuring the distances between the flanking regions and found that spontaneous distance outliers (i.e DNA breaks) appear more frequently at KRTAP-1 than at the representative common fragile site (CFS) FRA16D. Unlike CFSs, KRTAP-1 is not sensitive to aphidicolin. The exonuclease activity of DNA repair protein Mre11 protects KRTAP-1 from breaks, whereas CtIP does not. Breaks at KRTAP-1 lead to the palindromic duplication of the ERBB2 locus and trigger Breakage-Fusion-Bridge cycles. Our results indicate that an insufficiently investigated area of the human genome is fragile and could play a crucial role in cancer genome evolution.
Topics: Aphidicolin; Breast; Breast Neoplasms; Cells, Cultured; Chromosomal Instability; Chromosome Fragile Sites; DNA Breaks; DNA Copy Number Variations; DNA Repair; DNA, Neoplasm; Epithelial Cells; Female; Gene Amplification; Gene Duplication; Genes, erbB-2; Genetic Variation; Genomic Instability; Humans; Keratins, Hair-Specific; MRE11 Homologue Protein; Neoplasm Proteins; Whole Genome Sequencing
PubMed: 33290559
DOI: 10.1093/nar/gkaa1136 -
The Journal of Biological Chemistry Nov 2002Regulated gene expression is an important mechanism for controlling cell cycle progression in yeast and mammals, and genes involved in cell division-related processes...
Regulated gene expression is an important mechanism for controlling cell cycle progression in yeast and mammals, and genes involved in cell division-related processes often show transcriptional regulation dependent on cell cycle position. Analysis of cell cycle processes in plants has been hampered by the lack of synchronizable cell suspensions for Arabidopsis, and few cell cycle-regulated genes are known. Using a recently described synchrony system, we have analyzed RNA from sequential samples of Arabidopsis cells progressing through the cell cycle using Affymetrix Genearrays. We identify nearly 500 genes that robustly display significant fluctuation in expression, representing the first genomic analysis of cell cycle-regulated gene expression in any plant. In addition to the limited number of genes previously identified as cell cycle-regulated in plants, we also find specific patterns of regulation for genes known or suspected to be involved in signal transduction, transcriptional regulation, and hormonal regulation, including key genes of cytokinin response. Genes identified represent pathways that are cell cycle-regulated in other organisms and those involved in plant-specific processes. The range and number of cell cycle-regulated genes show the close integration of the plant cell cycle into a variety of cellular control and response pathways.
Topics: Aphidicolin; Arabidopsis; Cell Cycle; Cluster Analysis; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant
PubMed: 12169696
DOI: 10.1074/jbc.M207570200 -
Proceedings of the National Academy of... Feb 1983The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC...
The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase alpha has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the alpha-type polymerase: sensitivity to 0.2 M KCl, resistance to heat denaturation (45 degrees C for 15 min), an apparent Km of 5 microM for dATP, and an ability to copy poly(dT)X(rA)10 but not poly(rA)X(dT)12. The crude extracts and purified DNA polymerase alpha from the mutant cells are not inhibited by aphidicolin (greater than 0.6 microM). The apparent Km for dCTP with DNA polymerase alpha is 1.0 +/- 0.4 microM (mean +/- SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent Km for dCTP of 10 +/- 4 microM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.6.1.8) locus, is higher for mutant cells (42-73 x 10(-8)) than for parental cells (3-16 x 10(-8)). These data suggest a mechanism for aphidicolin resistance of the mutant--i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase alpha may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.
Topics: Animals; Aphidicolin; Cells, Cultured; Cricetinae; DNA Replication; DNA-Directed DNA Polymerase; Deoxyribonucleotides; Diterpenes; Drug Resistance; Kinetics; Mutation; Nucleic Acid Synthesis Inhibitors
PubMed: 6402775
DOI: 10.1073/pnas.80.3.797 -
Biochimica Et Biophysica Acta Dec 2013Muscle fructose 1,6-bisphosphate aldolase (ALDA) is a glycolytic enzyme which may localize both in nuclei and cytoplasm of cells, however its role in the nuclei is...
Muscle fructose 1,6-bisphosphate aldolase (ALDA) is a glycolytic enzyme which may localize both in nuclei and cytoplasm of cells, however its role in the nuclei is unclear. Here, we demonstrate the links between subcellular localization of ALDA and the cell cycle progression as well as the availability of energetic substrates. Results of our studies indicate that nuclear localization of ALDA correlates with the proliferative activity of the cells and with the expression of Ki-67, a marker of proliferation, both in the KLN-205 (mouse lung cancer cells) and human squamous cell lung cancer cells (hSCC). Chemically-induced block of cell cycle entry in S phase and the inhibition of transcription stimulate removal of ALDA from cells nuclei suggesting that nuclear ALDA is involved in cells proliferation. On the other hand, subcellular distribution of the enzyme also depends on the stress and pro-survival signals mediated by the Akt and the p38 pathways and, in non-proliferating cells, on the availability of glucose and lactate. The results presented here point to ALDA as a factor involved in the regulation of cells proliferation.
Topics: Animals; Aphidicolin; Cell Count; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Concanavalin A; Cytosol; Dactinomycin; Down-Regulation; Energy Metabolism; Fructose-Bisphosphate Aldolase; Humans; Ki-67 Antigen; Mice; Oligonucleotides, Antisense; Phosphorylation; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins c-akt; Rabbits; Resting Phase, Cell Cycle; Serum; p38 Mitogen-Activated Protein Kinases
PubMed: 23886627
DOI: 10.1016/j.bbamcr.2013.07.013 -
Nucleic Acids Research Sep 1991Highly compacted (40S) SV40 DNA replication intermediates formed in vivo during aphidicolin exposure and immediately broke down in two stages. In the rapid initial...
Highly compacted (40S) SV40 DNA replication intermediates formed in vivo during aphidicolin exposure and immediately broke down in two stages. In the rapid initial stage, single strand DNA breaks caused loss of superhelicity in the 40S replication intermediates. This DNA breakage was accompanied by the formation of strong, permanent protein-DNA crosslinks which reached a maximum as nicking of the aberrant DNA replication intermediates was completed. These protein-associated DNA strand breaks were not repaired. In the slower second stage of breakdown, the aberrant DNA replication intermediates remained nicked and strongly associated with protein as they underwent DNA replication fork breakage and recombinational changes to produce high molecular weight forms.
Topics: Aphidicolin; Chloroquine; DNA Replication; DNA, Viral; Electrophoresis, Gel, Two-Dimensional; Kinetics; Molecular Weight; Proteins; Simian virus 40; Virus Replication
PubMed: 1656388
DOI: 10.1093/nar/19.18.5065 -
Journal of Bacteriology Aug 1984Aphidicolin, a specific inhibitor of eucaryotic alpha DNA polymerase, inhibits the growth of halophilic arachaebacteria. In Halobacterium halobium, aphidicolin prevents...
Aphidicolin, a specific inhibitor of eucaryotic alpha DNA polymerase, inhibits the growth of halophilic arachaebacteria. In Halobacterium halobium, aphidicolin prevents cell division and DNA synthesis. These results suggest that arachaebacterial replicases are of the eucaryotic type.
Topics: Antiviral Agents; Aphidicolin; Bacterial Proteins; DNA Replication; Diterpenes; Halobacterium; Kinetics; RNA, Bacterial
PubMed: 6204969
DOI: 10.1128/jb.159.2.800-802.1984 -
Nucleic Acids Research Aug 2016Microsatellite DNAs that form non-B structures are implicated in replication fork stalling, DNA double strand breaks (DSBs) and human disease. Fanconi anemia (FA) is an...
Microsatellite DNAs that form non-B structures are implicated in replication fork stalling, DNA double strand breaks (DSBs) and human disease. Fanconi anemia (FA) is an inherited disorder in which mutations in at least nineteen genes are responsible for the phenotypes of genome instability and cancer predisposition. FA pathway proteins are active in the resolution of non-B DNA structures including interstrand crosslinks, G quadruplexes and DNA triplexes. In FANCJ helicase depleted cells, we show that hydroxyurea or aphidicolin treatment leads to loss of microsatellite polymerase chain reaction signals and to chromosome recombination at an ectopic hairpin forming CTG/CAG repeat in the HeLa genome. Moreover, diverse endogenous microsatellite signals were also lost upon replication stress after FANCJ depletion, and in FANCJ null patient cells. The phenotype of microsatellite signal instability is specific for FANCJ apart from the intact FA pathway, and is consistent with DSBs at microsatellites genome-wide in FANCJ depleted cells following replication stress.
Topics: Aphidicolin; Basic-Leucine Zipper Transcription Factors; Chromosomes, Human; DNA Replication; Fanconi Anemia; Fanconi Anemia Complementation Group D2 Protein; Fanconi Anemia Complementation Group Proteins; Gene Knockdown Techniques; Genome, Human; HeLa Cells; Humans; Microsatellite Repeats; Polymerase Chain Reaction; Recombination, Genetic; Stress, Physiological; Trinucleotide Repeat Expansion
PubMed: 27179029
DOI: 10.1093/nar/gkw433 -
Human Molecular Genetics Jul 2014Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Within the FA pathway, an upstream FA core complex mediates...
Fanconi anemia (FA) is a chromosome instability syndrome characterized by increased cancer predisposition. Within the FA pathway, an upstream FA core complex mediates monoubiquitination and recruitment of the central FANCD2 protein to sites of stalled replication forks. Once recruited, FANCD2 fulfills a dual role towards replication fork recovery: (i) it cooperates with BRCA2 and RAD51 to protect forks from nucleolytic degradation and (ii) it recruits the BLM helicase to promote replication fork restart while suppressing new origin firing. Intriguingly, FANCD2 and its interaction partners are also involved in homologous recombination (HR) repair of DNA double-strand breaks, hinting that FANCD2 utilizes HR proteins to mediate replication fork recovery. One such candidate is CtIP (CtBP-interacting protein), a key HR repair factor that functions in complex with BRCA1 and MRE11, but has not been investigated as putative player in the replication stress response. Here, we identify CtIP as a novel interaction partner of FANCD2. CtIP binds and stabilizes FANCD2 in a DNA damage- and FA core complex-independent manner, suggesting that FANCD2 monoubiquitination is dispensable for its interaction with CtIP. Following cellular treatment with a replication inhibitor, aphidicolin, FANCD2 recruits CtIP to transiently stalled, as well as collapsed, replication forks on chromatin. At stalled forks, CtIP cooperates with FANCD2 to promote fork restart and the suppression of new origin firing. Both functions are dependent on BRCA1 that controls the step-wise recruitment of MRE11, FANCD2 and finally CtIP to stalled replication forks, followed by their concerted actions to promote fork recovery.
Topics: Aphidicolin; BRCA1 Protein; Carrier Proteins; Cell Line; Chromatin; DNA Damage; DNA Replication; DNA-Binding Proteins; Endodeoxyribonucleases; Fanconi Anemia; Fanconi Anemia Complementation Group D2 Protein; Gene Expression Regulation; Humans; MRE11 Homologue Protein; Nuclear Proteins; Ubiquitination
PubMed: 24556218
DOI: 10.1093/hmg/ddu078