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ELife Oct 2023Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between...
Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged proliferating cell nuclear antigen (PCNA), a cell cycle marker and core component of the DNA replication machinery. We detected decreased chromatin mobility during the S-phase compared to G1 and G2 phases in tumor as well as normal diploid cells using automated particle tracking. To gain insight into the dynamical organization of the genome during DNA replication, we determined labeled chromatin domain sizes and analyzed their motion in replicating cells. By correlating chromatin mobility proximal to the active sites of DNA synthesis, we showed that chromatin motion was locally constrained at the sites of DNA replication. Furthermore, inhibiting DNA synthesis led to increased loading of DNA polymerases. This was accompanied by accumulation of the single-stranded DNA binding protein on the chromatin and activation of DNA helicases further restricting local chromatin motion. We, therefore, propose that it is the loading of replisomes but not their catalytic activity that reduces the dynamics of replicating chromatin segments in the S-phase as well as their accessibility and probability of interactions with other genomic regions.
Topics: Humans; Chromatin; DNA Replication; S Phase; Cell Cycle; DNA Helicases
PubMed: 37906089
DOI: 10.7554/eLife.87572 -
The Journal of General Virology Oct 2023The baculovirus IE1 gene encodes a multifunctional protein that is essential for both DNA replication and RNA transcription of the virus. Prior to viral DNA replication,...
The baculovirus IE1 gene encodes a multifunctional protein that is essential for both DNA replication and RNA transcription of the virus. Prior to viral DNA replication, IE1 promotes early gene transcription when localized in -dependent foci. During viral DNA replication, the IE1 foci expand and fuse to generate the virogenic stroma (VS) with IE1 found in the VS reticulum. To explore the IE1 structural features essential for this coordinated localization, we constructed various IE1 mutants based on three putative domains (N, I, and C). We determined that a BDI motif located in the intrinsic disorder region (IDR) between the N and I domains acts as a nuclear localization signal, whereas BDII and HLH in the C domain are required for VS localization in infected cells or for chromosomal association in uninfected mitotic cells. Deletion of the SLiM (short linear motif) located in the I domain restrains both nuclear- and VS localization. Intra-molecular fluorescence resonance energy transfer (FRET) probes of IE1 mutants revealed a conformational change of the I-C two-domain fragment during infection, which was inhibited by aphidicolin, suggesting that IE1 undergoes a stage-dependent conformational change. Further, homo-dimerization of the I domain and stage-dependent conformational changes require an intact SLiM. Mutational analysis of SLiM revealed that VS localization and chromosomal association were retained following S291A and S291E substitutions, but -dependent focus formation differed between the two mutations. These results suggest that coordinated IE1 localization is controlled by SLiM-dependent conformational changes that are potentially switched by the phosphorylation state of the SLiM.
Topics: Baculoviridae; DNA Replication; Virus Replication; DNA, Viral; Phosphorylation
PubMed: 37877992
DOI: 10.1099/jgv.0.001910 -
Emerging Topics in Life Sciences Dec 2023Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and...
Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and constrictions on metaphase chromosomes under mild replication stress. These replication-sensitive CFS regions are preferentially unstable during cancer development, as reflected by their association with copy number variants (CNVs) frequently arise in most tumor types. Over the years, it became clear that a combination of different characteristics underlies the enhanced sensitivity of CFSs to replication stress. As of today, there is a strong evidence that the core fragility regions along CFSs overlap with actively transcribed large genes with delayed replication timing upon replication stress. Recently, the mechanistic basis for CFS instability was further extended to regions which span topologically associated domain (TAD) boundaries, generating a fragility signature composed of replication, transcription and genome organization. The presence of difficult-to-replicate AT-rich repeats was one of the early features suggested to characterize a subgroup of CFSs. These long stretches of AT-dinucleotide have the potential to fold into stable secondary structures which may impede replication fork progression, leaving the region under-replicated. Here, we focus on the molecular mechanisms underlying repeat instability at CFSs and on the proteins involved in the resolution of secondary structure impediments arising along repetitive sequence elements which are essential for the maintenance of genome stability.
Topics: Humans; DNA Replication; Chromosome Fragile Sites; DNA Replication Timing; Chromosomal Instability; DNA
PubMed: 37876349
DOI: 10.1042/ETLS20230023 -
Phytochemistry Dec 2023Natural products that inhibit cell cycle progression may have potential as anticancer agents. In this study, cell cycle inhibition of microbial culture extracts was...
Natural products that inhibit cell cycle progression may have potential as anticancer agents. In this study, cell cycle inhibition of microbial culture extracts was screened by fluorescent images using HeLa/Fucci2 cells. The culture extract of a fungus, Akanthomyces sp., inhibited the cell cycle progression at the S/G2/M phases, and bioassay-guided fractionation of the extract afforded three previously undescribed aphidicolin derivatives, namely akanthomins A-C, and an undescribed chromone glycoside, specifically 9-hydroxyeugenetin 9-O-β-d-(4-O-methyl)glucopyranoside, in addition to aphidicolin. The chemical structures of these compounds were elucidated by spectroscopic analysis and chemical derivatization. Using a flow cytometer, akanthomin A and aphidicolin were found to inhibit cell cycle progression at the S phase.
Topics: Aphidicolin; Cell Cycle; Cell Division; Hypocreales; Plant Extracts
PubMed: 37806468
DOI: 10.1016/j.phytochem.2023.113885 -
Life Sciences Nov 2023Studies in the past have shown that inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase sensitizes cancer cells to genotoxic anticancer treatments,...
AIMS
Studies in the past have shown that inhibition of the ataxia telangiectasia and Rad3-related (ATR) kinase sensitizes cancer cells to genotoxic anticancer treatments, however, clinical use of ATR inhibitors in combination with DNA damaging chemotherapy is limited due to toxicity in healthy tissues. In this study, we investigated the synergistic anticancer effect between ATR inhibition and oxidative DNA damage induced by the thioredoxin reductase inhibitor auranofin.
MAIN METHODS
Cytotoxicity was evaluated by cell viability assays. Western blot, comet assay, immunostaining and flow cytometry were performed to dissect the underlying mechanisms. In vivo efficacy was examined against tumor xenografts.
KEY FINDINGS
Nontoxic doses of auranofin alone increased the levels of reactive oxygen species (ROS) in cancer but not noncancerous cells, resulting in oxidative DNA damage and activation of the ATR DNA damage response pathway selectively in cancer cells. Inhibition of ATR in auranofin-treated cancer cells resulted in unscheduled firing of dormant DNA replication origins, abrogation of the S phase cell cycle checkpoint and extensive DNA breakage, leading to replication catastrophe and potent synergistic lethality. Both the antioxidant NAC and the DNA polymerase inhibitor aphidicolin reduced replication stress and synergistic cytotoxicity, implicating replication stress-driven catastrophic cell death resulted from collision between oxidative DNA damage and dysregulated DNA replication. In vivo, auranofin and VE822 coadministration enabled marked regressions of tumor xenografts, while each drug alone had no effect.
SIGNIFICANCE
As increased generation of ROS is a universal feature of tumors, our findings may open new routes to broaden the therapeutic potential of ATR inhibitors.
Topics: Humans; Auranofin; Reactive Oxygen Species; DNA Damage; Neoplasms; Oxidative Stress; Protein Kinase Inhibitors; DNA; Checkpoint Kinase 1; Cell Line, Tumor; Ataxia Telangiectasia Mutated Proteins
PubMed: 37778414
DOI: 10.1016/j.lfs.2023.122131 -
Chromosome Research : An International... Aug 2023Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural...
Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2'-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.
Topics: Humans; Induced Pluripotent Stem Cells; Aphidicolin; Caffeine; Chromosomes, Human, Pair 12; In Situ Hybridization, Fluorescence; Intracellular Signaling Peptides and Proteins
PubMed: 37597021
DOI: 10.1007/s10577-023-09729-5 -
Journal of Physiology and Pharmacology... Apr 2023Building a precise alternative neurotoxicological test is of great importance to respond to societal and ethical requirements. In this study, a new developmental...
Building a precise alternative neurotoxicological test is of great importance to respond to societal and ethical requirements. In this study, a new developmental neurotoxicity test (DNT) was established with the human neural progenitor cell line. ReNcell CX cells were exposed to neurotoxic chemicals (aphidicolin, hydroxyurea, cytosine arabinoside, 5-fluorouracil, and ochratoxin A) or non-neurotoxic chemicals (sodium gluconate, sodium bicarbonate, penicillin G, and saccharin). Propidium iodide (PI) was used to evaluate cell viability. BrdU and Ki-76 were employed to determine cell proliferation. Based on the cell viability and proliferation, mathematical models were built by linear discriminant analysis. Furthermore, the neurotoxic-considered chemicals inhibited cell cycle progression at the protein level, supporting the biomolecular rationale for the predictive model. Overall, these results show that the new test method can be used to determine the potential developmental neurotoxicants or new drug candidates.
Topics: Humans; Ki-67 Antigen; Neural Stem Cells; Neurotoxicity Syndromes; Cell Line
PubMed: 37453095
DOI: 10.26402/jpp.2023.2.07 -
Genetics May 2023Chromosome breakage plays an important role in the evolution of karyotypes and can produce deleterious effects within a single individual, such as aneuploidy or cancer....
Chromosome breakage plays an important role in the evolution of karyotypes and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not fully understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes in Drosophila melanogaster, we find that breakage under tension also tends to occur in specific hotspots. Our experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. We analyzed the breakage patterns of 3 different ring-X chromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all 3 chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, we found that the hotspot locations are not conserved between the 3 chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility. Additionally, the frequency of dicentric breakage and the durability of each chromosome's spindle attachment vary significantly between the 3 chromosomes and are correlated with the origin of the centromere and the amount of pericentric heterochromatin. We suggest that different centromere strengths could account for this.
Topics: Animals; Humans; Chromosome Breakage; Drosophila melanogaster; Heterochromatin; Centromere; X Chromosome
PubMed: 37010100
DOI: 10.1093/genetics/iyad052 -
Nature Communications Mar 2023Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show...
Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.
Topics: Humans; Reactive Oxygen Species; DNA Replication; S Phase; DNA-Binding Proteins; Hydroxyurea; DNA
PubMed: 36997515
DOI: 10.1038/s41467-023-37341-y -
Life Science Alliance Apr 2023Eukaryotic genomes are duplicated from thousands of replication origins that fire sequentially forming a defined spatiotemporal pattern of replication clusters. The...
Eukaryotic genomes are duplicated from thousands of replication origins that fire sequentially forming a defined spatiotemporal pattern of replication clusters. The temporal order of DNA replication is determined by chromatin architecture and, more specifically, by chromatin contacts that are stabilized by RIF1. Here, we show that RIF1 localizes near newly synthesized DNA. In cells exposed to the DNA replication inhibitor aphidicolin, suppression of RIF1 markedly decreased the efficacy of isolation of proteins on nascent DNA, suggesting that the isolation of proteins on nascent DNA procedure is biased by chromatin topology. RIF1 was required to limit the accumulation of DNA lesions induced by aphidicolin treatment and promoted the recruitment of cohesins in the vicinity of nascent DNA. Collectively, the data suggest that the stabilization of chromatin topology by RIF1 limits replication-associated genomic instability.
Topics: Chromatin; Aphidicolin; Telomere-Binding Proteins; DNA; DNA Replication
PubMed: 36746532
DOI: 10.26508/lsa.202101186