Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 49.528
Filtrar
1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445496

RESUMO

Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin-CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina/metabolismo , Animais , Replicação do DNA , Humanos , Mitose , Processamento de Proteína Pós-Traducional
2.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445553

RESUMO

Since their discovery, R-loops have been associated with both physiological and pathological functions that are conserved across species. R-loops are a source of replication stress and genome instability, as seen in neurodegenerative disorders and cancer. In response, cells have evolved pathways to prevent R-loop accumulation as well as to resolve them. A growing body of evidence correlates R-loop accumulation with changes in the epigenetic landscape. However, the role of chromatin modification and remodeling in R-loops homeostasis remains unclear. This review covers various mechanisms precluding R-loop accumulation and highlights the role of chromatin modifiers and remodelers in facilitating timely R-loop resolution. We also discuss the enigmatic role of RNA:DNA hybrids in facilitating DNA repair, epigenetic landscape and the potential role of replication fork preservation pathways, active fork stability and stalled fork protection pathways, in avoiding replication-transcription conflicts. Finally, we discuss the potential role of several Chro-Mates (chromatin modifiers and remodelers) in the likely differentiation between persistent/detrimental R-loops and transient/benign R-loops that assist in various physiological processes relevant for therapeutic interventions.


Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , Reparo do DNA , Replicação do DNA , Instabilidade Genômica , Estruturas R-Loop , Humanos
3.
Nat Microbiol ; 6(9): 1175-1187, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373624

RESUMO

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle.


Assuntos
Proteínas de Bactérias/metabolismo , Ciclo Celular , Proteínas do Citoesqueleto/metabolismo , Replicação do DNA , Streptococcus pneumoniae/citologia , Streptococcus pneumoniae/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , Streptococcus pneumoniae/genética
4.
Nat Commun ; 12(1): 4841, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404770

RESUMO

RAS proteins are GTPases that lie upstream of a signaling network impacting cell fate determination. How cells integrate RAS activity to balance proliferation and cellular senescence is still incompletely characterized. Here, we identify ZNF768 as a phosphoprotein destabilized upon RAS activation. We report that ZNF768 depletion impairs proliferation and induces senescence by modulating the expression of key cell cycle effectors and established p53 targets. ZNF768 levels decrease in response to replicative-, stress- and oncogene-induced senescence. Interestingly, ZNF768 overexpression contributes to bypass RAS-induced senescence by repressing the p53 pathway. Furthermore, we show that ZNF768 interacts with and represses p53 phosphorylation and activity. Cancer genomics and immunohistochemical analyses reveal that ZNF768 is often amplified and/or overexpressed in tumors, suggesting that cells could use ZNF768 to bypass senescence, sustain proliferation and promote malignant transformation. Thus, we identify ZNF768 as a protein linking oncogenic signaling to the control of cell fate decision and proliferation.


Assuntos
Senescência Celular/genética , Genes ras/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Carcinogênese , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Transformação Celular Neoplásica , Replicação do DNA , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genômica , Células HeLa , Humanos , Oncogenes , Fenótipo , Fosfoproteínas , Fosforilação , Repressão Psicológica , Transdução de Sinais , Proteínas ras/genética
5.
Nat Microbiol ; 6(9): 1108-1109, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373625
6.
Int J Mol Sci ; 22(15)2021 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-34360694

RESUMO

Termination of DNA replication, the final stage of genome duplication, is surprisingly complex, and failures to bring DNA synthesis to an accurate conclusion can impact genome stability and cell viability. In Escherichia coli, termination takes place in a specialised termination area opposite the origin. A 'replication fork trap' is formed by unidirectional fork barriers via the binding of Tus protein to genomic ter sites. Such a fork trap system is found in some bacterial species, but it appears not to be a general feature of bacterial chromosomes. The biochemical properties of fork trap systems have been extensively characterised, but little is known about their precise physiological roles. In this study, we compare locations and distributions of ter terminator sites in E. coli genomes across all phylogenetic groups, including Shigella. Our analysis shows that all ter sites are highly conserved in E. coli, with slightly more variability in the Shigella genomes. Our sequence analysis of ter sites and Tus proteins shows that the fork trap is likely to be active in all strains investigated. In addition, our analysis shows that the dif chromosome dimer resolution site is consistently located between the innermost ter sites, even if rearrangements have changed the location of the innermost termination area. Our data further support the idea that the replication fork trap has an important physiological role that provides an evolutionary advantage.


Assuntos
Cromossomos Bacterianos , Replicação do DNA , Escherichia coli/genética , Filogenia , Sequências Reguladoras de Ácido Nucleico/genética , DNA Bacteriano , Enterobacteriaceae/genética
7.
Science ; 373(6558): 1030-1035, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34385354

RESUMO

Biological mechanisms underlying human germline mutations remain largely unknown. We statistically decompose variation in the rate and spectra of mutations along the genome using volume-regularized nonnegative matrix factorization. The analysis of a sequencing dataset (TOPMed) reveals nine processes that explain the variation in mutation properties between loci. We provide a biological interpretation for seven of these processes. We associate one process with bulky DNA lesions that are resolved asymmetrically with respect to transcription and replication. Two processes track direction of replication fork and replication timing, respectively. We identify a mutagenic effect of active demethylation primarily acting in regulatory regions and a mutagenic effect of long interspersed nuclear elements. We localize a mutagenic process specific to oocytes from population sequencing data. This process appears transcriptionally asymmetric.


Assuntos
Genoma Humano , Mutação em Linhagem Germinativa , Algoritmos , Ilhas de CpG , Dano ao DNA , Desmetilação do DNA , Análise Mutacional de DNA , Replicação do DNA , Variação Genética , Células Germinativas , Humanos , Elementos Nucleotídeos Longos e Dispersos , Mutagênese , Oócitos/fisiologia , Transcrição Genética
8.
Front Cell Infect Microbiol ; 11: 670564, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34422676

RESUMO

Trypanosomatids are a group of primitive unicellular eukaryotes that can cause diseases in plants, insects, animals, and humans. Kinetoplast genome integrity is key to trypanosomatid cell survival and viability. Kinetoplast DNA (kDNA) is usually under attack by reactive oxygen and nitric species (ROS and RNS), damaging the DNA, and the cells must remove and repair those oxidatively generated lesions in order to survive and proliferate. Base excision repair (BER) is a well-conserved pathway for DNA repair after base damage, single-base loss, and single-strand breaks, which can arise from ROS, RSN, environmental genotoxic agents, and UV irradiation. A powerful BER system has been described in the T. cruzi kinetoplast and it is mainly carried out by DNA polymerase ß (pol ß) and DNA polymerase ß-PAK (pol ß-PAK), which are kinetoplast-located in T. cruzi as well as in other trypanosomatids. Both pol ß and pol ß-PAK belong to the X-family of DNA polymerases (pol X family), perform BER in trypanosomatids, and display intrinsic 5-deoxyribose phosphate (dRP) lyase and DNA polymerase activities. However, only Pol ß-PAK is able to carry out trans-lesion synthesis (TLS) across 8oxoG lesions. T. cruzi cells overexpressing pol ß are more resistant to ROS and are also more efficient to repair 8oxoG compared to control cells. Pol ß seems to play a role in kDNA replication, since it associates with kinetoplast antipodal sites in those development stages in trypanosomatids which are competent for cell replication. ROS treatment of cells induces the overexpression of pol ß, indicating that plays a role in kDNA repair. In this review, we will summarize the main features of trypanosomatid minicircle kDNA replication and the biochemical characteristics of pol ß-like enzymes and their involvement in BER and kDNA replication. We also summarize key structural features of trypanosomatid pol ß compared to their mammalian (human) counterpart.


Assuntos
DNA Polimerase beta , Animais , DNA , Dano ao DNA , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Reparo do DNA , Replicação do DNA , Humanos
9.
Appl Microbiol Biotechnol ; 105(14-15): 5959-5972, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34357429

RESUMO

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.


Assuntos
Origem de Replicação , Yarrowia , Centrômero , Replicação do DNA , Engenharia Metabólica , Plasmídeos/genética , Yarrowia/genética
10.
Mol Cell ; 81(14): 2873-2874, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34270943

RESUMO

Wang et al. (2021) comprehensively map DNA replication initiation events across the human genome using single-molecule optical resolution mapping and find that initiation events are randomly distributed across broad initiation zones that are only utilized in a stochastic fashion across a population of cells.


Assuntos
Genoma Humano , Origem de Replicação , Replicação do DNA , Genoma Humano/genética , Humanos , Origem de Replicação/genética
11.
Mol Cell ; 81(15): 3128-3144.e7, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34216544

RESUMO

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions.


Assuntos
Proteína BRCA1/genética , Replicação do DNA/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Linhagem Celular , Cisplatino/farmacologia , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos Endogâmicos NOD , RNA Helicases/genética , Rad51 Recombinase/genética , Proteína de Replicação A/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
12.
Mol Cell ; 81(15): 3110-3127.e14, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34233157

RESUMO

SPT6 is a histone chaperone that tightly binds RNA polymerase II (RNAPII) during transcription elongation. However, its primary role in transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in RNAPII behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for RNAPII processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in RNAPII termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the prolonged absence of SPT6. In conclusion, targeted degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and RNAPII elongation factor.


Assuntos
RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo , Linhagem Celular , Replicação do DNA , Humanos , Ácidos Indolacéticos/farmacologia , Poliadenilação , Proteólise/efeitos dos fármacos , RNA/biossíntese , RNA Polimerase II/genética , Fatores de Transcrição/genética
13.
Nat Commun ; 12(1): 4531, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312383

RESUMO

Recent developments in synthetic biology may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. Here, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this Perspective, we discuss potential paths towards an integrated synthetic cell cycle.


Assuntos
Células Artificiais , Mimetismo Biológico/genética , Ciclo Celular/genética , Replicação do DNA/genética , Modelos Genéticos , Biologia Sintética/métodos , Bacteriófagos/genética , Escherichia coli/genética , Biossíntese de Proteínas/genética , Biologia Sintética/tendências , Transcrição Genética/genética
14.
Nat Commun ; 12(1): 4373, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272385

RESUMO

Although homologous recombination (HR) is indicated as a high-fidelity repair mechanism, break-induced replication (BIR), a subtype of HR, is a mutagenic mechanism that leads to chromosome rearrangements. It remains poorly understood how cells suppress mutagenic BIR. Trapping of Topoisomerase 1 by camptothecin (CPT) in a cleavage complex on the DNA can be transformed into single-ended double-strand breaks (seDSBs) upon DNA replication or colliding with transcriptional machinery. Here, we demonstrate a role of Abraxas in limiting seDSBs undergoing BIR-dependent mitotic DNA synthesis. Through counteracting K63-linked ubiquitin modification, Abraxas restricts SLX4/Mus81 recruitment to CPT damage sites for cleavage and subsequent resection processed by MRE11 endonuclease, CtIP, and DNA2/BLM. Uncontrolled SLX4/MUS81 loading and excessive end resection due to Abraxas-deficiency leads to increased mitotic DNA synthesis via RAD52- and POLD3- dependent, RAD51-independent BIR and extensive chromosome aberrations. Our work implicates Abraxas/BRCA1-A complex as a critical regulator that restrains BIR for protection of genome stability.


Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Recombinases/metabolismo , Animais , Camptotecina/farmacologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , DNA Polimerase III/metabolismo , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Recombinação Homóloga , Humanos , Proteína Homóloga a MRE11/metabolismo , Camundongos , RNA Interferente Pequeno , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Inibidores da Topoisomerase I/farmacologia , Ubiquitinação
15.
Mol Cell ; 81(14): 2989-3006.e9, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34197737

RESUMO

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Replicação do DNA/genética , DNA/genética , Exonucleases/genética , Instabilidade Genômica/genética , RecQ Helicases/genética , Linhagem Celular , Linhagem Celular Tumoral , Dano ao DNA/genética , DNA Helicases/genética , Análise Mutacional de DNA/métodos , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Células HeLa , Humanos , Mutação/genética , Oncogenes/genética , Fosforilação/genética , Regulação para Cima/genética
16.
Nucleic Acids Res ; 49(13): 7492-7506, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197599

RESUMO

Tumor heterogeneity includes variable and fluctuating oxygen concentrations, which result in the accumulation of hypoxic regions in most solid tumors. Tumor hypoxia leads to increased therapy resistance and has been linked to genomic instability. Here, we tested the hypothesis that exposure to levels of hypoxia that cause replication stress could increase APOBEC activity and the accumulation of APOBEC-mediated mutations. APOBEC-dependent mutational signatures have been well-characterized, although the physiological conditions which underpin them have not been described. We demonstrate that fluctuating/cyclic hypoxic conditions which lead to replication catastrophe induce the expression and activity of APOBEC3B. In contrast, stable/chronic hypoxic conditions which induce replication stress in the absence of DNA damage are not sufficient to induce APOBEC3B. Most importantly, the number of APOBEC-mediated mutations in patient tumors correlated with a hypoxia signature. Together, our data support the conclusion that hypoxia-induced replication catastrophe drives genomic instability in tumors, specifically through increasing the activity of APOBEC3B.


Assuntos
Citidina Desaminase/metabolismo , Replicação do DNA , Antígenos de Histocompatibilidade Menor/metabolismo , Neoplasias/enzimologia , Desaminases APOBEC/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Desaminação , Humanos , Hidroxiureia/toxicidade , Estresse Fisiológico/genética
17.
Nucleic Acids Res ; 49(13): 7537-7553, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197600

RESUMO

The synaptonemal complex (SC) is a proteinaceous structure that mediates homolog engagement and genetic recombination during meiosis. In budding yeast, Zip-Mer-Msh (ZMM) proteins promote crossover (CO) formation and initiate SC formation. During SC elongation, the SUMOylated SC component Ecm11 and the Ecm11-interacting protein Gmc2 facilitate the polymerization of Zip1, an SC central region component. Through physical recombination, cytological, and genetic analyses, we found that ecm11 and gmc2 mutants exhibit chromosome-specific defects in meiotic recombination. CO frequencies on a short chromosome (chromosome III) were reduced, whereas CO and non-crossover frequencies on a long chromosome (chromosome VII) were elevated. Further, in ecm11 and gmc2 mutants, more double-strand breaks (DSBs) were formed on a long chromosome during late prophase I, implying that the Ecm11-Gmc2 (EG) complex is involved in the homeostatic regulation of DSB formation. The EG complex may participate in joint molecule (JM) processing and/or double-Holliday junction resolution for ZMM-dependent CO-designated recombination. Absence of the EG complex ameliorated the JM-processing defect in zmm mutants, suggesting a role for the EG complex in suppressing ZMM-independent recombination. Our results suggest that the SC central region functions as a compartment for sequestering recombination-associated proteins to regulate meiosis specificity during recombination.


Assuntos
Proteínas de Ciclo Celular/genética , Troca Genética , Quebras de DNA de Cadeia Dupla , Meiose/genética , Proteínas de Saccharomyces cerevisiae/genética , Complexo Sinaptonêmico/metabolismo , Cromossomos Fúngicos , Replicação do DNA , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Retroalimentação Fisiológica , Deleção de Genes , Recombinação Genética , Saccharomyces cerevisiae/genética , Temperatura , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética
18.
Nucleic Acids Res ; 49(13): 7318-7329, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197604

RESUMO

Integrating omics data with quantification of biological traits provides unparalleled opportunities for discovery of genetic regulators by in silico inference. However, current approaches to analyze genetic-perturbation screens are limited by their reliance on annotation libraries for prioritization of hits and subsequent targeted experimentation. Here, we present iTARGEX (identification of Trait-Associated Regulatory Genes via mixture regression using EXpectation maximization), an association framework with no requirement of a priori knowledge of gene function. After creating this tool, we used it to test associations between gene expression profiles and two biological traits in single-gene deletion budding yeast mutants, including transcription homeostasis during S phase and global protein turnover. For each trait, we discovered novel regulators without prior functional annotations. The functional effects of the novel candidates were then validated experimentally, providing solid evidence for their roles in the respective traits. Hence, we conclude that iTARGEX can reliably identify novel factors involved in given biological traits. As such, it is capable of converting genome-wide observations into causal gene function predictions. Further application of iTARGEX in other contexts is expected to facilitate the discovery of new regulators and provide observations for novel mechanistic hypotheses regarding different biological traits and phenotypes.


Assuntos
Perfilação da Expressão Gênica , Genes Reguladores , Proteólise , Fase S/genética , Software , Transcrição Genética , Proteínas de Transporte/genética , Biologia Computacional/métodos , Replicação do DNA , Deleção de Genes , Homeostase , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
Nucleic Acids Res ; 49(13): 7476-7491, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197614

RESUMO

Poly (ADP-ribose) polymerase inhibitor (PARPi)-based therapies initially reduce tumor burden but eventually lead to acquired resistance in cancer patients with BRCA1 or BRCA2 mutation. To understand the potential PARPi resistance mechanisms, we performed whole-genome CRISPR screens to discover genetic alterations that change the gene essentiality in cells with inducible depletion of BRCA2. We identified that several RNA Polymerase II transcription Mediator complex components, especially Cyclin C (CCNC) as synthetic survival targets upon BRCA2 loss. Total mRNA sequencing demonstrated that loss of CCNC could activate the transforming growth factor (TGF)-beta signaling pathway and extracellular matrix (ECM)-receptor interaction pathway, however the inhibition of these pathways could not reverse cell survival in BRCA2 depleted CCNC-knockout cells, indicating that the activation of these pathways is not required for the resistance. Moreover, we showed that the improved survival is not due to restoration of homologous recombination repair although decreased DNA damage signaling was observed. Interestingly, loss of CCNC could restore replication fork stability in BRCA2 deficient cells, which may contribute to PARPi resistance. Taken together, our data reveal CCNC as a critical genetic determinant upon BRCA2 loss of function, which may help the development of novel therapeutic strategies that overcome PARPi resistance.


Assuntos
Proteína BRCA2/genética , Ciclina C/genética , Proteína BRCA2/metabolismo , Sistemas CRISPR-Cas , Sobrevivência Celular , Dano ao DNA , Replicação do DNA , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Complexo Mediador/genética , Complexo Mediador/fisiologia , Reparo de DNA por Recombinação , Estresse Fisiológico/genética
20.
J Theor Biol ; 527: 110822, 2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34214567

RESUMO

It is likely that RNA replication began non-enzymatically, and that polymerases were later selected to speed up the process. We consider replication mechanisms in modern viruses and ask which of these is possible non-enzymatically, using mathematical models and experimental data found in the literature to estimate rates of RNA synthesis and replication. Replication via alternating plus and minus strands is found in some single-stranded RNA viruses. However, if this occurred non-enzymatically it would lead to double-stranded RNA that would not separate. With some form of environmental cycling, such as temperature, salinity, or pH cycling, double-stranded RNA can be melted to form single-stranded RNA, although re-annealing of existing strands would then occur much faster than synthesis of new strands. We show that re-annealing blocks this form of replication at a very low concentration of strands. Other kinds of viruses synthesize linear double strands from single strands and then make new single strands from double strands via strand-displacement. This does not require environmental cycling and is not blocked by re-annealing. However, under non-enzymatic conditions, if strand-displacement occurs from a linear template, we expect the incomplete new strand to be almost always displaced by the tail end of the old strand through toehold-mediated displacement. A third kind of replication in viruses and viroids is rolling-circle replication which occurs via strand-displacement on a circular template. Rolling-circle replication does not require environmental cycling and is not prevented by toehold-mediated displacement. Rolling-circle replication is therefore expected to occur non-enzymatically and is a likely starting point for the evolution of polymerase-catalysed replication.


Assuntos
Replicação do DNA , Recombinação Genética , RNA
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...