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1.
Dev Growth Differ ; 66(7): 384-393, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39305158

RESUMO

The development of new technologies opens new avenues in the research field. Gene knockout is a key method for analyzing gene function in mice. Currently, conditional gene knockout strategies are employed to examine temporal and spatial gene function. However, phenotypes are sometimes not observed because of the time required for depletion due to the long half-life of the target proteins. Protein knockdown using an improved auxin-inducible degron system, AID2, overcomes such difficulties owing to rapid and efficient target depletion. We observed depletion of AID-tagged proteins within a few to several hours by a simple intraperitoneal injection of the auxin analog, 5-Ph-IAA, which is much shorter than the time required for target depletion using conditional gene knockout. Importantly, the loss of protein is reversible, making protein knockdown useful to measure the effects of transient loss of protein function. Here, we also established several mouse lines useful for AID2-medicated protein knockdown, which include knock-in mouse lines in the ROSA26 locus; one expresses TIR1(F74G), and the other is the reporter expressing AID-mCherry. We also established a germ-cell-specific TIR1 line and confirmed the protein knockdown specificity. In addition, we introduced an AID tag to an endogenous protein, DCP2 via the CAS9-mediated gene editing method. We confirmed that the protein was effectively eliminated by TIR1(F74G), which resulted in the similar phenotype observed in knockout mouse within 20 h.


Assuntos
Ácidos Indolacéticos , Animais , Camundongos , Ácidos Indolacéticos/farmacologia , Ácidos Indolacéticos/metabolismo , Proteólise/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Degrons
2.
Nat Commun ; 15(1): 7152, 2024 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-39169041

RESUMO

For accurate mitotic cell division, replicated chromatin must be assembled into chromosomes and faithfully segregated into daughter cells. While protein factors like condensin play key roles in this process, it is unclear how chromosome assembly proceeds as molecular events of nucleosomes in living cells and how condensins act on nucleosomes to organize chromosomes. To approach these questions, we investigate nucleosome behavior during mitosis of living human cells using single-nucleosome tracking, combined with rapid-protein depletion technology and computational modeling. Our results show that local nucleosome motion becomes increasingly constrained during mitotic chromosome assembly, which is functionally distinct from condensed apoptotic chromatin. Condensins act as molecular crosslinkers, locally constraining nucleosomes to organize chromosomes. Additionally, nucleosome-nucleosome interactions via histone tails constrain and compact whole chromosomes. Our findings elucidate the physical nature of the chromosome assembly process during mitosis.


Assuntos
Adenosina Trifosfatases , Cromatina , Proteínas de Ligação a DNA , Mitose , Complexos Multiproteicos , Nucleossomos , Humanos , Nucleossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Complexos Multiproteicos/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Células HeLa , Cromossomos Humanos/metabolismo , Cromossomos Humanos/genética , Cromossomos/metabolismo
3.
EMBO Rep ; 25(9): 4062-4077, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39179892

RESUMO

Acute protein knockdown is a powerful approach to dissecting protein function in dynamic cellular processes. We previously reported an improved auxin-inducible degron system, AID2, but recently noted that its ability to induce degradation of some essential replication factors, such as ORC1 and CDC6, was not enough to induce lethality. Here, we present combinational degron technologies to control two proteins or enhance target depletion. For this purpose, we initially compare PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrate control of cohesin and condensin with AID2 and BromoTag, respectively. We develop a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrate that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we show that co-depletion of ORC1 and CDC6 by the double-degron system completely suppresses DNA replication, and the cells enter mitosis with single-chromatid chromosomes, indicating that DNA replication is uncoupled from cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses.


Assuntos
Adenosina Trifosfatases , Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Ligação a DNA , Complexos Multiproteicos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Complexos Multiproteicos/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Reconhecimento de Origem/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Técnicas de Silenciamento de Genes , Coesinas , Mitose/efeitos dos fármacos , Mitose/genética , Proteólise , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Degrons
4.
bioRxiv ; 2024 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-39026740

RESUMO

Enhancers are key drivers of gene regulation thought to act via 3D physical interactions with the promoters of their target genes. However, genome-wide depletions of architectural proteins such as cohesin result in only limited changes in gene expression, despite a loss of contact domains and loops. Consequently, the role of cohesin and 3D contacts in enhancer function remains debated. Here, we developed CRISPRi of regulatory elements upon degron operation (CRUDO), a novel approach to measure how changes in contact frequency impact enhancer effects on target genes by perturbing enhancers with CRISPRi and measuring gene expression in the presence or absence of cohesin. We systematically perturbed all 1,039 candidate enhancers near five cohesin-dependent genes and identified 34 enhancer-gene regulatory interactions. Of 26 regulatory interactions with sufficient statistical power to evaluate cohesin dependence, 18 show cohesin-dependent effects. A decrease in enhancer-promoter contact frequency upon removal of cohesin is frequently accompanied by a decrease in the regulatory effect of the enhancer on gene expression, consistent with a contact-based model for enhancer function. However, changes in contact frequency and regulatory effects on gene expression vary as a function of distance, with distal enhancers (e.g., >50Kb) experiencing much larger changes than proximal ones (e.g., <50Kb). Because most enhancers are located close to their target genes, these observations can explain how only a small subset of genes - those with strong distal enhancers - are sensitive to cohesin. Together, our results illuminate how 3D contacts, influenced by both cohesin and genomic distance, tune enhancer effects on gene expression.

5.
DNA Repair (Amst) ; 141: 103713, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38959715

RESUMO

Eukaryotic DNA replication is a tightly controlled process that occurs in two main steps, i.e., licensing and firing, which take place in the G1 and S phases of the cell cycle, respectively. In Saccharomyces cerevisiae, the budding yeast, replication origins contain consensus sequences that are recognized and bound by the licensing factor Orc1-6, which then recruits the replicative Mcm2-7 helicase. By contrast, mammalian initiation sites lack such consensus sequences, and the mammalian ORC does not exhibit sequence specificity. Studies performed over the past decades have identified replication initiation sites in the mammalian genome using sequencing-based assays, raising the question of whether replication initiation occurs at confined sites or in broad zones across the genome. Although recent reports have shown that the licensed MCMs in mammalian cells are broadly distributed, suggesting that ORC-dependent licensing may not determine the initiation sites/zones, they are predominantly located upstream of actively transcribed genes. This review compares the mechanism of replication initiation in yeast and mammalian cells, summarizes the sequencing-based technologies used for the identification of initiation sites/zones, and proposes a possible mechanism of initiation-site/zone selection in mammalian cells. Future directions and challenges in this field are also discussed.


Assuntos
Replicação do DNA , Origem de Replicação , Saccharomyces cerevisiae , Animais , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Reconhecimento de Origem/genética , Mamíferos/genética , Genoma , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/genética
6.
iScience ; 27(7): 110260, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-39055910

RESUMO

To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for "backup" mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes.

7.
bioRxiv ; 2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38585954

RESUMO

Disrupted nuclear shape is associated with multiple pathological processes including premature aging disorders, cancer-relevant chromosomal rearrangements, and DNA damage. Nuclear blebs (i.e., herniations of the nuclear envelope) have been induced by (1) nuclear compression, (2) nuclear migration (e.g., cancer metastasis), (3) actin contraction, (4) lamin mutation or depletion, and (5) heterochromatin enzyme inhibition. Recent work has shown that chromatin transformation is a hallmark of bleb formation, but the transformation of higher-order structures in blebs is not well understood. As higher-order chromatin has been shown to assemble into nanoscopic packing domains, we investigated if (1) packing domain organization is altered within nuclear blebs and (2) if alteration in packing domain structure contributed to bleb formation. Using Dual-Partial Wave Spectroscopic microscopy, we show that chromatin packing domains within blebs are transformed both by B-type lamin depletion and the inhibition of heterochromatin enzymes compared to the nuclear body. Pairing these results with single-molecule localization microscopy of constitutive heterochromatin, we show fragmentation of nanoscopic heterochromatin domains within bleb domains. Overall, these findings indicate that translocation into blebs results in a fragmented higher-order chromatin structure. SUMMARY STATEMENT: Nuclear blebs are linked to various pathologies, including cancer and premature aging disorders. We investigate alterations in higher-order chromatin structure within blebs, revealing fragmentation of nanoscopic heterochromatin domains.

8.
Nat Commun ; 15(1): 2960, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38580649

RESUMO

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.


Assuntos
Metilação de DNA , Neoplasias , Humanos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Neoplasias/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Genome Biol ; 25(1): 77, 2024 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-38519987

RESUMO

BACKGROUND: B-type lamins are critical nuclear envelope proteins that interact with the three-dimensional genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron technology. RESULTS: Using live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, Stochastic Optical Reconstruction Microscopy (STORM), in situ Hi-C, CRISPR-Sirius, and fluorescence in situ hybridization (FISH), we demonstrate that lamin B1 and lamin B2 are critical structural components of the nuclear periphery that create a repressive compartment for peripheral-associated genes. Lamin B1 and lamin B2 depletion minimally alters higher-order chromatin folding but disrupts cell morphology, significantly increases chromatin mobility, redistributes both constitutive and facultative heterochromatin, and induces differential gene expression both within and near lamin-associated domain (LAD) boundaries. Critically, we demonstrate that chromatin territories expand as upregulated genes within LADs radially shift inwards. Our results indicate that the mechanism of action of B-type lamins comes from their role in constraining chromatin motion and spatial positioning of gene-specific loci, heterochromatin, and chromatin domains. CONCLUSIONS: Our findings suggest that, while B-type lamin degradation does not significantly change genome topology, it has major implications for three-dimensional chromatin conformation at the single-cell level both at the lamina-associated periphery and the non-LAD-associated nuclear interior with concomitant genome-wide transcriptional changes. This raises intriguing questions about the individual and overlapping roles of lamin B1 and lamin B2 in cellular function and disease.


Assuntos
Cromatina , Lamina Tipo B , Animais , Lamina Tipo B/genética , Heterocromatina , Hibridização in Situ Fluorescente , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Laminas , Expressão Gênica , Mamíferos/genética
10.
Nat Commun ; 15(1): 981, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38302485

RESUMO

Despite drastic cellular changes during cleavage, a mitotic spindle assembles in each blastomere to accurately segregate duplicated chromosomes. Mechanisms of mitotic spindle assembly have been extensively studied using small somatic cells. However, mechanisms of spindle assembly in large vertebrate embryos remain little understood. Here, we establish functional assay systems in medaka (Oryzias latipes) embryos by combining CRISPR knock-in with auxin-inducible degron technology. Live imaging reveals several unexpected features of microtubule organization and centrosome positioning that achieve rapid, accurate cleavage. Importantly, Ran-GTP assembles a dense microtubule network at the metaphase spindle center that is essential for chromosome segregation in early embryos. This unique spindle structure is remodeled into a typical short, somatic-like spindle after blastula stages, when Ran-GTP becomes dispensable for chromosome segregation. We propose that despite the presence of centrosomes, the chromosome-derived Ran-GTP pathway has essential roles in functional spindle assembly in large, rapidly dividing vertebrate early embryos, similar to acentrosomal spindle assembly in oocytes.


Assuntos
Oryzias , Animais , Oryzias/genética , Segregação de Cromossomos , Centrossomo/metabolismo , Fuso Acromático/metabolismo , Microtúbulos/metabolismo , Vertebrados , Guanosina Trifosfato/metabolismo , Mitose
11.
Genes Cells ; 29(1): 39-51, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37963657

RESUMO

The c-Jun N-terminal kinase-associated leucine zipper protein (JLP), a scaffold protein of mitogen-activated protein kinase signaling pathways, is a multifunctional protein involved in a variety of cellular processes. It has been reported that JLP is overexpressed in various types of cancer and is expected to be a potential therapeutic target. However, whether and how JLP overexpression affects non-transformed cells remain unknown. Here, we aimed to investigate the effect of JLP overexpression on chromosomal stability in human non-transformed cells and the mechanisms involved. We found that aneuploidy was induced in JLP-overexpressed cells. Moreover, we established JLP-inducible cell lines and observed an increased frequency of chromosome missegregation, reduced time from nuclear envelope breakdown to anaphase onset, and decreased levels of the spindle assembly checkpoint (SAC) components at the prometaphase kinetochore in cells overexpressing the wild-type JLP. In contrast, we observed that a point mutant JLP lacking the ability to interact with dynein light intermediate chain 1 (DLIC1) failed to induce chromosomal instability. Our results suggest that overexpression of the wild-type JLP facilitates premature SAC silencing through interaction with DLIC1, leading to aneuploidy. This study provides a novel insight into the mechanism through which JLP overexpression is associated with cancer development and progression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Neoplasias , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Zíper de Leucina , Dineínas/genética , Dineínas/metabolismo , Neoplasias/metabolismo , Instabilidade Cromossômica , Aneuploidia , Mitose
12.
Nat Commun ; 14(1): 5071, 2023 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-37604812

RESUMO

Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication stress) is a major cause of cancer, premature aging and a spectrum of other human disorders. Over the years, TRAIP E3 ubiquitin ligase has been shown to play a role in various cellular processes that govern genome integrity and faultless segregation. TRAIP is essential for cell viability, and mutations in TRAIP ubiquitin ligase activity lead to primordial dwarfism in patients. Here, we have determined the mechanism of inhibition of cell proliferation in TRAIP-depleted cells. We have taken advantage of the auxin induced degron system to rapidly degrade TRAIP within cells and to dissect the importance of various functions of TRAIP in different stages of the cell cycle. We conclude that upon rapid TRAIP degradation, specifically in S-phase, cells cease to proliferate, arrest in G2 stage of the cell cycle and undergo senescence. Our findings reveal that TRAIP works in S-phase to prevent DNA damage at transcription start sites, caused by replication-transcription conflicts.


Assuntos
Ubiquitina-Proteína Ligases , Humanos , Fase S/genética , Divisão Celular/genética , Proliferação de Células/genética , Ciclo Celular , Sobrevivência Celular , Ubiquitina-Proteína Ligases/genética
13.
Science ; 381(6664): eadi3448, 2023 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-37590370

RESUMO

CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery.


Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Ligação a DNA , Proteínas de Manutenção de Minicromossomo , Proteínas Nucleares , Animais , Humanos , Camundongos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Mapeamento de Interação de Proteínas/métodos , Simulação por Computador , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nanismo/genética , Microcefalia/genética , Xenopus laevis
14.
bioRxiv ; 2023 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-37425796

RESUMO

BACKGROUND: B-type lamins are critical nuclear envelope proteins that interact with the 3D genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron (AID) technology. RESULTS: Paired with a suite of novel technologies, live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, in situ Hi-C, and CRISPR-Sirius, we demonstrate that lamin B1 and lamin B2 depletion transforms chromatin mobility, heterochromatin positioning, gene expression, and loci-positioning with minimal disruption to mesoscale chromatin folding. Using the AID system, we show that the disruption of B-lamins alters gene expression both within and outside lamin associated domains, with distinct mechanistic patterns depending on their localization. Critically, we demonstrate that chromatin dynamics, positioning of constitutive and facultative heterochromatic markers, and chromosome positioning near the nuclear periphery are significantly altered, indicating that the mechanism of action of B-type lamins is derived from their role in maintaining chromatin dynamics and spatial positioning. CONCLUSIONS: Our findings suggest that the mechanistic role of B-type lamins is stabilization of heterochromatin and chromosomal positioning along the nuclear periphery. We conclude that degrading lamin B1 and lamin B2 has several functional consequences related to both structural disease and cancer.

15.
Nature ; 620(7972): 209-217, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37438531

RESUMO

The human genome functions as a three-dimensional chromatin polymer, driven by a complex collection of chromosome interactions1-3. Although the molecular rules governing these interactions are being quickly elucidated, relatively few proteins regulating this process have been identified. Here, to address this gap, we developed high-throughput DNA or RNA labelling with optimized Oligopaints (HiDRO)-an automated imaging pipeline that enables the quantitative measurement of chromatin interactions in single cells across thousands of samples. By screening the human druggable genome, we identified more than 300 factors that influence genome folding during interphase. Among these, 43 genes were validated as either increasing or decreasing interactions between topologically associating domains. Our findings show that genetic or chemical inhibition of the ubiquitous kinase GSK3A leads to increased long-range chromatin looping interactions in a genome-wide and cohesin-dependent manner. These results demonstrate the importance of GSK3A signalling in nuclear architecture and the use of HiDRO for identifying mechanisms of spatial genome organization.


Assuntos
Cromatina , Posicionamento Cromossômico , Cromossomos Humanos , Genoma Humano , Quinases da Glicogênio Sintase , Ensaios de Triagem em Larga Escala , Análise de Célula Única , Humanos , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Posicionamento Cromossômico/efeitos dos fármacos , Cromossomos Humanos/efeitos dos fármacos , Cromossomos Humanos/genética , Cromossomos Humanos/metabolismo , DNA/análise , DNA/metabolismo , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Quinases da Glicogênio Sintase/antagonistas & inibidores , Quinases da Glicogênio Sintase/deficiência , Quinases da Glicogênio Sintase/genética , Ensaios de Triagem em Larga Escala/métodos , Interfase , Reprodutibilidade dos Testes , RNA/análise , RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Célula Única/métodos , Coesinas
16.
Biochem Soc Trans ; 51(3): 1289-1295, 2023 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-37145026

RESUMO

The mini-chromosome maintenance proteins 2-7 (MCM2-7) hexamer is a protein complex that is key for eukaryotic DNA replication, which occurs only once per cell cycle. To achieve DNA replication, eukaryotic cells developed multiple mechanisms that control the timing of the loading of the hexamer onto chromatin and its activation as the replicative helicase. MCM2-7 is highly abundant in proliferating cells, which confers resistance to replication stress. Thus, the presence of an excess of MCM2-7 is important for maintaining genome integrity. However, the mechanism via which high MCM2-7 levels are achieved, other than the transcriptional upregulation of the MCM genes in the G1 phase, remained unknown. Recently, we and others reported that the MCM-binding protein (MCMBP) plays a role in the maintenance of high MCM2-7 levels and hypothesized that MCMBP functions as a chaperone in the assembly of the MCM2-7 hexamer. In this review, we discuss the roles of MCMBP in the control of MCM proteins and propose a model of the assembly of the MCM2-7 hexamer. Furthermore, we discuss a potential mechanism of the licensing checkpoint, which arrests the cells in the G1 phase when the levels of chromatin-bound MCM2-7 are reduced, and the possibility of targeting MCMBP as a chemotherapy for cancer.


Assuntos
Proteínas de Ciclo Celular , Proteínas de Manutenção de Minicromossomo , Proteínas de Manutenção de Minicromossomo/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Cromatina , Replicação do DNA
17.
Science ; 380(6643): 382-387, 2023 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-37104614

RESUMO

Replication fork reversal safeguards genome integrity as a replication stress response. DNA translocases and the RAD51 recombinase catalyze reversal. However, it remains unknown why RAD51 is required and what happens to the replication machinery during reversal. We find that RAD51 uses its strand exchange activity to circumvent the replicative helicase, which remains bound to the stalled fork. RAD51 is not required for fork reversal if the helicase is unloaded. Thus, we propose that RAD51 creates a parental DNA duplex behind the helicase that is used as a substrate by the DNA translocases for branch migration to create a reversed fork structure. Our data explain how fork reversal happens while maintaining the helicase in a position poised to restart DNA synthesis and complete genome duplication.


Assuntos
Replicação do DNA , Proteínas de Ligação a DNA , Rad51 Recombinase , Proteínas de Transporte/metabolismo , DNA/genética , DNA/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Humanos , Células HCT116 , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Xenopus
18.
Nat Commun ; 14(1): 706, 2023 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-36759509

RESUMO

Oncogene activation creates DNA replication stress (RS) in cancer cells, which can generate under-replicated DNA regions (UDRs) that persist until cells enter mitosis. UDRs also have the potential to generate DNA bridges in anaphase cells or micronuclei in the daughter cells, which could promote genomic instability. To suppress such damaging changes to the genome, human cells have developed a strategy to conduct 'unscheduled' DNA synthesis in mitosis (termed MiDAS) that serves to rescue under-replicated loci. Previous studies have shown that MiDAS proceeds via a POLD3-dependent pathway that shows some features of break-induced replication. Here, we define how human cells utilize both DNA gap filling (REV1 and Pol ζ) and replicative (Pol δ) DNA polymerases to complete genome duplication following a perturbed S-phase. We present evidence for the existence of a polymerase-switch during MiDAS that is required for new DNA synthesis at UDRs. Moreover, we reveal that, upon oncogene activation, cancer cell survival is significantly compromised when REV1 is depleted, suggesting that REV1 inhibition might be a feasible approach for the treatment of some human cancers.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Reparo do DNA , DNA/genética , DNA/metabolismo , Dano ao DNA
19.
Drug Discov Ther ; 17(1): 10-17, 2023 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-36642508

RESUMO

The extracellular signal-regulated kinase (ERK) 1 and 2 intracellular signaling pathways play key roles in a variety of cellular processes, such as proliferation and differentiation. Dysregulation of ERK1/2 signaling has been implicated in many diseases, including cancer. Although ERK1/2 signaling pathways have been extensively studied, controversy remains as to whether ERK1 and ERK2 have specific or redundant functions. In this study, we examined the functional roles of ERK1 and ERK2 in cell proliferation and cell cycle progression using an auxin-inducible degron system combined with gene knockout technology. We found that ERK1/2 double depletion, but not ERK1 or ERK2 depletion, substantially inhibited the proliferation of HCT116 cells during G1-S transition. We further demonstrated that ERK1/2-double-depleted cells were much more tolerant to etoposide-induced G2/M arrest than ERK1 or ERK2 single-knockout cells. Together, these results strongly suggest the functional redundancy of ERK1 and ERK2 in both the G1-S transition under physiological conditions and the DNA damage-induced G2/M checkpoint. Our findings substantially advance understanding of the ERK1/2 pathways, which could have strong implications for future pharmacological developments.


Assuntos
Apoptose , MAP Quinases Reguladas por Sinal Extracelular , Humanos , Etoposídeo , Células HCT116 , Pontos de Checagem da Fase G2 do Ciclo Celular , Linhagem Celular Tumoral , Fosforilação
20.
Commun Biol ; 5(1): 1420, 2022 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-36577784

RESUMO

Cellular senescence caused by oncogenic stimuli is associated with the development of various age-related pathologies through the senescence-associated secretory phenotype (SASP). SASP is mediated by the activation of cytoplasmic nucleic acid sensors. However, the molecular mechanism underlying the accumulation of nucleotide ligands in senescent cells is unclear. In this study, we revealed that the expression of RNaseH2A, which removes ribonucleoside monophosphates (rNMPs) from the genome, is regulated by E2F transcription factors, and it decreases during cellular senescence. Residual rNMPs cause genomic DNA fragmentation and aberrant activation of cytoplasmic nucleic acid sensors, thereby provoking subsequent SASP factor gene expression in senescent cells. In addition, RNaseH2A expression was significantly decreased in aged mouse tissues and cells from individuals with Werner syndrome. Furthermore, RNaseH2A degradation using the auxin-inducible degron system induced the accumulation of nucleotide ligands and induction of certain tumourigenic SASP-like factors, promoting the metastatic properties of colorectal cancer cells. Our results indicate that RNaseH2A downregulation provokes SASP through nucleotide ligand accumulation, which likely contributes to the pathological features of senescent, progeroid, and cancer cells.


Assuntos
DNA , Neoplasias , Animais , Camundongos , Senescência Celular/genética , Fragmentação do DNA , Regulação para Baixo , Expressão Gênica , Genômica , Ligantes , Neoplasias/genética , Neoplasias/metabolismo , Nucleotídeos , Fenótipo , Humanos , Linhagem Celular
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