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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 -
The EMBO Journal Feb 1995Tissue homeostasis and the prevention of neoplasia require regulatory co-ordination between cellular proliferation and apoptosis. Several cellular proteins, including...
Tissue homeostasis and the prevention of neoplasia require regulatory co-ordination between cellular proliferation and apoptosis. Several cellular proteins, including c-myc and E2F, as well as viral proteins such as E1A, have dual functions as positive regulators of apoptosis and proliferation. The product of the retinoblastoma tumor suppressor gene, pRb, binds these proteins and is known to function in growth suppression. To examine whether pRb may function as a negative regulator of both proliferation and apoptosis, we analyzed apoptosis induced in transfected derivatives of the human osteosarcoma cell line SAOS-2. Ionizing radiation induced apoptosis in a time- and dose-dependent manner in SAOS-2 cells, which lack pRb expression. In both a transient and stable transfection assay, SAOS-2 derivatives expressing wild-type (wt) pRb exhibited increased viability and decreased apoptosis following treatment at a variety of radiation doses. Expression in SAOS-2 of a mutant pRb that fails to complex with several known binding partners of pRb, including E1A and E2F, did not protect SAOS-2 cells from apoptosis. Radiation exposure induced a G2 arrest in SAOS-2 and in derivatives expressing pRb. Inhibition of DNA synthesis and cell cycle progression by aphidicolin treatment failed to protect SAOS-2 cells or pRb-expressing isolates from undergoing apoptosis. Our data document a novel function for pRb in suppressing apoptosis and suggest that several proteins shown to induce apoptosis, including E1A, E2F and c-myc, may do so by interfering with the protective function of pRb.
Topics: Aphidicolin; Apoptosis; Cell Cycle; Dose-Response Relationship, Radiation; Genes, Retinoblastoma; Humans; Osteosarcoma; Radiation Tolerance; Retinoblastoma Protein; Transfection; Tumor Cells, Cultured; X-Rays
PubMed: 7859736
DOI: 10.1002/j.1460-2075.1995.tb07022.x -
Nucleic Acids Research Jul 2009FRAXA is one of a number of fragile sites in human chromosomes that are induced by agents like fluorodeoxyuridine (FdU) that affect intracellular thymidylate levels....
FRAXA is one of a number of fragile sites in human chromosomes that are induced by agents like fluorodeoxyuridine (FdU) that affect intracellular thymidylate levels. FRAXA coincides with a >200 CGG*CCG repeat tract in the 5' UTR of the FMR1 gene, and alleles prone to fragility are associated with Fragile X (FX) syndrome, one of the leading genetic causes of intellectual disability. Using siRNA depletion, we show that ATR is involved in protecting the genome against FdU-induced chromosome fragility. We also show that FdU increases the number of gamma-H2AX foci seen in both normal and patient cells and increases the frequency with which the FMR1 gene colocalizes with these foci in patient cells. In the presence of FdU and KU55933, an ATM inhibitor, the incidence of chromosome fragility is reduced, suggesting that ATM contributes to FdU-induced chromosome fragility. Since both ATR and ATM are involved in preventing aphidicolin-sensitive fragile sites, our data suggest that the lesions responsible for aphidicolin-induced and FdU-induced fragile sites differ. FRAXA also displays a second form of chromosome fragility in absence of FdU, which our data suggest is normally prevented by an ATM-dependent process.
Topics: Aphidicolin; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Line; Chromosome Breakage; Chromosome Fragile Sites; Chromosome Fragility; DNA Damage; DNA Repair; DNA-Binding Proteins; Floxuridine; Fragile X Mental Retardation Protein; Fragile X Syndrome; Gene Knockdown Techniques; Histones; Humans; Male; Morpholines; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrones; Staurosporine; Tumor Suppressor Proteins
PubMed: 19465392
DOI: 10.1093/nar/gkp391 -
Nucleic Acids Research Apr 2018During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as...
During mild replication stress provoked by low dose aphidicolin (APH) treatment, the key Fanconi anemia protein FANCD2 accumulates on common fragile sites, observed as sister foci, and protects genome stability. To gain further insights into FANCD2 function and its regulatory mechanisms, we examined the genome-wide chromatin localization of FANCD2 in this setting by ChIP-seq analysis. We found that FANCD2 mostly accumulates in the central regions of a set of large transcribed genes that were extensively overlapped with known CFS. Consistent with previous studies, we found that this FANCD2 retention is R-loop-dependent. However, FANCD2 monoubiquitination and RPA foci formation were still induced in cells depleted of R-loops. Interestingly, we detected increased Proximal Ligation Assay dots between FANCD2 and R-loops following APH treatment, which was suppressed by transcriptional inhibition. Collectively, our data suggested that R-loops are required to retain FANCD2 in chromatin at the middle intronic region of large genes, while the replication stress-induced upstream events leading to the FA pathway activation are not triggered by R-loops.
Topics: Aphidicolin; Cell Line, Tumor; Chromatin; Chromosome Fragile Sites; DNA; DNA Damage; DNA Repair; DNA Replication; Enzyme Inhibitors; Fanconi Anemia Complementation Group D2 Protein; Gene Expression Regulation; Genomic Instability; Humans; Nucleic Acid Conformation; Signal Transduction; Ubiquitination
PubMed: 29394375
DOI: 10.1093/nar/gky058 -
Antimicrobial Agents and Chemotherapy Jun 1983A plaque-reduction assay was used to examine the susceptibility of five phosphonoformic acid-resistant variants of herpes simplex virus type 1 to arabinosylnucleosides...
A plaque-reduction assay was used to examine the susceptibility of five phosphonoformic acid-resistant variants of herpes simplex virus type 1 to arabinosylnucleosides and aphidicolin. These viruses were cross-resistant to arabinosylhypoxanthine and to arabinosyladenine when tested in the absence of deoxycoformycin, a deaminase inhibitor. In the presence of deoxycoformycin, no cross-resistance between arabinosyladenine and phosphonoformic acid was observed. The two variants tested were cross-resistant to arabinosylthymine, and all five variants were collaterally susceptible to aphidicolin inhibition.
Topics: Antiviral Agents; Aphidicolin; Arabinonucleosides; Coformycin; DNA-Directed DNA Polymerase; Diterpenes; Drug Resistance, Microbial; Foscarnet; Pentostatin; Phosphonoacetic Acid; Simplexvirus; Thymidine; Thymidine Kinase; Vidarabine
PubMed: 6311091
DOI: 10.1128/AAC.23.6.914 -
PloS One 2013Fragile site breakage was previously shown to result in rearrangement of the RET oncogene, resembling the rearrangements found in thyroid cancer. Common fragile sites...
Fragile site breakage was previously shown to result in rearrangement of the RET oncogene, resembling the rearrangements found in thyroid cancer. Common fragile sites are specific regions of the genome with a high susceptibility to DNA breakage under conditions that partially inhibit DNA replication, and often coincide with genes deleted, amplified, or rearranged in cancer. While a substantial amount of work has been performed investigating DNA repair and cell cycle checkpoint proteins vital for maintaining stability at fragile sites, little is known about the initial events leading to DNA breakage at these sites. The purpose of this study was to investigate these initial events through the detection of aphidicolin (APH)-induced DNA breakage within the RET oncogene, in which 144 APH-induced DNA breakpoints were mapped on the nucleotide level in human thyroid cells within intron 11 of RET, the breakpoint cluster region found in patients. These breakpoints were located at or near DNA topoisomerase I and/or II predicted cleavage sites, as well as at DNA secondary structural features recognized and preferentially cleaved by DNA topoisomerases I and II. Co-treatment of thyroid cells with APH and the topoisomerase catalytic inhibitors, betulinic acid and merbarone, significantly decreased APH-induced fragile site breakage within RET intron 11 and within the common fragile site FRA3B. These data demonstrate that DNA topoisomerases I and II are involved in initiating APH-induced common fragile site breakage at RET, and may engage the recognition of DNA secondary structures formed during perturbed DNA replication.
Topics: Aphidicolin; Base Sequence; Cell Line; DNA; DNA Breaks; DNA Replication; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Humans; Introns; Molecular Sequence Data; Nucleic Acid Conformation; Oncogenes; Proteolysis; Proto-Oncogene Proteins c-ret; Topoisomerase Inhibitors
PubMed: 24040417
DOI: 10.1371/journal.pone.0075741 -
Plant Signaling & Behavior 2014Cell cycle is an essential process in growth and development of living organisms consists of the replication and mitotic phases separated by 2 gap phases; G1 and G2. It...
Cell cycle is an essential process in growth and development of living organisms consists of the replication and mitotic phases separated by 2 gap phases; G1 and G2. It is tightly controlled at the molecular level and especially at the level of transcription. Precise regulation of the cell cycle is of central significance for plant growth and development and transcription factors are global regulators of gene expression playing essential roles in cell cycle regulation. This study has uncovered TFs that are involved in the control of cell cycle progression. With the aid of multi-parallel quantitative RT-PCR, the expression changes of 1880 TFs represented in the Arabidopsis TF platform was monitored in Arabidopsis synchronous MM2d cells during a 19 h period representing different time points corresponding to the 4 cell cycle phases after treatment of MM2d cells with Aphidicolin. Comparative TF expression analyses performed on synchronous cells resulted in the identification of 239 TFs differentially expressed during the cell cycle, while about one third of TFs were constitutively expressed through all time points. Phase-specific TFs were also identified.
Topics: Aphidicolin; Arabidopsis; Cell Cycle; Cell Proliferation; Cells, Cultured; Cluster Analysis; Down-Regulation; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Plant; Genes, Plant; Suspensions; Transcription Factors; Up-Regulation
PubMed: 25482767
DOI: 10.4161/15592316.2014.972864 -
MBio May 2017The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon...
The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a -acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter.
Topics: Aphidicolin; DNA Replication; DNA, Viral; Gene Expression Regulation, Viral; Genes, Viral; Heterogeneous-Nuclear Ribonucleoproteins; Host-Pathogen Interactions; Human papillomavirus 18; Humans; Keratinocytes; Promoter Regions, Genetic; RNA Splicing; Replication Origin; Transcription, Genetic; Virus Replication
PubMed: 28559488
DOI: 10.1128/mBio.00713-17 -
Life Science Alliance Apr 2019We have shown previously that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in the S-phase is driven through...
We have shown previously that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in the S-phase is driven through polyubiquitylation of one of the replicative helicase subunits (Mcm7) by Cul2 ubiquitin ligase. Interestingly, upon inhibition of this pathway in embryos, the replisomes retained on chromatin were unloaded in the subsequent mitosis. Here, we show that this mitotic replisome disassembly pathway exists in egg extract and we determine the first elements of its regulation. The mitotic disassembly pathway depends on the formation of K6- and K63-linked ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and the activity of p97/VCP protein segregase. Unlike in lower eukaryotes, however, it does not require SUMO modifications. Importantly, we also show that this process can remove all replisomes from mitotic chromatin, including stalled ones, which indicates a wide application for this pathway over being just a "backup" for terminated replisomes. Finally, we characterise the composition of the replisome retained on chromatin until mitosis.
Topics: Animals; Aphidicolin; Caffeine; Chromatin; Cyclins; Cyclopentanes; DNA Replication; Female; Male; Minichromosome Maintenance Complex Component 7; Mitosis; Ovum; Pyrimidines; Small Ubiquitin-Related Modifier Proteins; Spermatozoa; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitination; Valosin Containing Protein; Xenopus Proteins; Xenopus laevis
PubMed: 30979826
DOI: 10.26508/lsa.201900390 -
Scientific Reports Nov 2019Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and - as a consequence - rates of biochemical reactions in a cell. Nanoviscosity is an...
Nanoviscosity of the cytoplasm is a key factor affecting diffusion of biomolecules and - as a consequence - rates of biochemical reactions in a cell. Nanoviscosity is an outcome of variable chemical and structural factors, which can temporarily change with cell-cycle associated changes of intracellular architecture. Thus, the question arises, whether rates of biochemical reactions depend on the point of cell cycle. In this paper we address this topic by constant observation of nanoviscosity of HeLa cells cytoplasm during S, G2 and G1 phases after Aphidicolin synchronization. For this purpose we measured diffusion rates of EGFP molecules using fluorescence correlation spectroscopy (FCS). To our surprise, a counter-intuitive stability of cytoplasmic viscosity was observed during the cell cycle. Our results hint at possible existence of robust mechanism maintaining stable physiological viscosity of the cytoplasm, despite huge structural changes during cell cycle.
Topics: Aphidicolin; Biophysical Phenomena; Cell Cycle; Cell Size; Cytoplasm; Flow Cytometry; HeLa Cells; Humans; Viscosity
PubMed: 31712575
DOI: 10.1038/s41598-019-52758-6