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1.
Methods Cell Biol ; 182: 199-219, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38359977

RESUMO

Transcription-replication conflicts (TRCs) represent a potent endogenous source of replication stress. Besides the spatial and temporal coordination of replication and transcription programs, cells employ many additional mechanisms to resolve TRCs in a timely manner, thereby avoiding replication fork stalling and genomic instability. Proximity ligation assays (PLA) using antibodies against actively elongating RNA Polymerase II (RNAPIIpS2) and PCNA to detect proximity (<40nm) between transcribing RNA polymerases and replication forks can be used to assess and quantify TRC levels in cells. A complementary fluorescence microscopy approach to assess the spatial coordination of transcription and replication activities in the nucleus is to quantify the colocalization (200-400nm) between active transcription and ongoing replication using immunofluorescence staining with an antibody against elongating RNA Polymerase II (RNAPIIpS2) and EdU-Click-it pulse-labelling, respectively. Despite significant efforts to automate image analysis, the need for manual verification, correction, and complementation of automated processes creates a bottleneck for efficient, high-throughput and large-scale imaging. Here, we describe an automated Fiji image analysis macro that allows the user to automate the measurement of RNAPIIpS2 and EdU levels and extract the key parameters such as transcription-replication (TR) colocalization and TRC-PLA foci count from single cells in a high throughput manner. While we showcase the usability of this analysis pipeline for quantifying TR colocalization and TRC-PLA in mouse embryonic stem cells (mESCs), the analysis pipeline is designed as a generally applicable tool allowing the quantification of nuclear signals, colocalization and foci count in various model systems and cell types.


Assuntos
Replicação do DNA , RNA Polimerase II , Animais , Camundongos , RNA Polimerase II/genética , Replicação do DNA/genética , Mamíferos
2.
Nucleic Acids Res ; 51(22): 12303-12324, 2023 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-37956271

RESUMO

Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci. MATAC-Seq relies on preferential modification of accessible DNA by methyltransferases combined with Nanopore-Sequencing for direct readout of methylated DNA-bases. Applying MATAC-Seq to selected early-efficient and late-inefficient yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an accessible nucleosome-free region in combination with well-positioned +1 and +2 nucleosomes as a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during eukaryotic DNA replication. Consequently, our single-molecule chromatin accessibility assay will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.


Assuntos
Origem de Replicação , Saccharomyces cerevisiae , Cromatina/genética , DNA , Replicação do DNA , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
3.
Cell Rep ; 42(2): 112045, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36701236

RESUMO

The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to known origin interactors, we find the microtubule-binding Ask1/DASH complex as an origin-regulating factor. Strikingly, tethering of Ask1 to individual origin sites advances replication timing (RT) of the targeted chromosomal domain. Targeted degradation of Ask1 globally changes RT of a subset of origins, which can be reproduced by inhibiting microtubule dynamics. Thus, our findings mechanistically connect RT and chromosomal organization via Ask1/DASH with the microtubule cytoskeleton.


Assuntos
Proteínas Associadas aos Microtúbulos , Origem de Replicação , Proteínas de Saccharomyces cerevisiae , Cromatina/metabolismo , DNA/metabolismo , Replicação do DNA , Período de Replicação do DNA , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , Proteômica , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Life (Basel) ; 11(7)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209204

RESUMO

Transcription-replication conflicts occur when the two critical cellular machineries responsible for gene expression and genome duplication collide with each other on the same genomic location. Although both prokaryotic and eukaryotic cells have evolved multiple mechanisms to coordinate these processes on individual chromosomes, it is now clear that conflicts can arise due to aberrant transcription regulation and premature proliferation, leading to DNA replication stress and genomic instability. As both are considered hallmarks of aging and human diseases such as cancer, understanding the cellular consequences of conflicts is of paramount importance. In this article, we summarize our current knowledge on where and when collisions occur and how these encounters affect the genome and chromatin landscape of cells. Finally, we conclude with the different cellular pathways and multiple mechanisms that cells have put in place at conflict sites to ensure the resolution of conflicts and accurate genome duplication.

5.
J Mol Biol ; 432(15): 4232-4243, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32084415

RESUMO

In eukaryotes, telomeres are repetitive sequences at the end of chromosomes, which are maintained in a constitutive heterochromatin state. It is now known that telomeres can be actively transcribed, leading to the production of a telomeric repeat-containing noncoding RNA called TERRA. Due to its sequence complementarity to the telomerase template, it was suggested early on that TERRA could be an inhibitor of telomerase. Since then, TERRA has been shown to be involved in heterochromatin formation at telomeres, to invade telomeric dsDNA and form R-loops, and even to promote telomerase recruitment at short telomeres. All these functions depend on the diverse capacities of this lncRNA to bind various cofactors, act as a scaffold, and promote higher-order complexes in cells. In this review, it will be highlighted as to how these properties of TERRA work together to regulate telomerase activity at telomeres.


Assuntos
RNA Longo não Codificante/genética , Telomerase/metabolismo , Telômero/metabolismo , Animais , Regulação Enzimológica da Expressão Gênica , Humanos , Estruturas R-Loop , RNA Longo não Codificante/química , Telômero/química
6.
Curr Genet ; 64(5): 1117-1127, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29569051

RESUMO

Telomeres are maintained in a heterochromatic state that represses transcription of subtelomeric genes, a phenomenon known as telomere position effect. Nevertheless, telomeric DNA is actively transcribed, leading to the synthesis of telomeric repeat-containing noncoding RNA or TERRA. This nuclear noncoding RNA has been proposed to play important roles at telomeres, regulating their silencing, capping, repair and elongation by telomerase. In the budding yeast Saccharomyces cerevisiae, TERRA accumulation is repressed by telomeric silencing and the Rat1 exonuclease. On the other hand, telomere shortening promotes expression of TERRA. So far, little is known about the biological processes that induce TERRA expression in yeast. Understanding the dynamics of TERRA expression and localization is essential to define its function in telomere biology. Here, we aim to study the dynamics of TERRA expression during yeast cell growth. Using live-cell imaging, RNA-FISH and quantitative RT-PCR, we show that TERRA expression is induced as yeast cells undergo diauxic shift, a lag phase during which yeast cells switch their metabolism from anaerobic fermentation to oxidative respiration. This induction is transient as TERRA levels decrease during post-diauxic shift. The increased expression of TERRA is not due to the shortening of telomeres or increased stability of this transcript. Surprisingly, this induction is coincident with a cytoplasmic accumulation of TERRA molecules. Our results suggest that TERRA transcripts may play extranuclear functions with important implications in telomere biology and add a novel layer of complexity in the interplay between telomere biology, metabolism and stress response.


Assuntos
RNA Fúngico/metabolismo , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae/genética , Telômero/genética , Transporte Biológico , Divisão Celular , Citoplasma/metabolismo , Hibridização in Situ Fluorescente , RNA Fúngico/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Reação em Cadeia da Polimerase em Tempo Real , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico , Transcrição Gênica
7.
Methods Mol Biol ; 1672: 387-402, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29043638

RESUMO

Telomerase, the enzyme that elongates telomeres in most eukaryotes, is a ribonucleoprotein complex composed of a reverse transcriptase catalytic subunit (TERT in human, Est2 in the budding yeast S. cerevisiae), regulatory factors and a noncoding RNA called hTERC (in human) or TLC1 (in budding yeast). Telomerase trafficking is a major process in the biogenesis and regulation of telomerase action at telomeres. Due to its higher signal-to-noise ratio, imaging of the telomerase RNA moiety is frequently used to determine telomerase intracellular localization. Here we describe how to image telomerase RNA in human and yeast cells using fluorescence in situ hybridization.


Assuntos
Hibridização in Situ Fluorescente , Imagem Molecular , RNA/genética , Saccharomycetales/genética , Telomerase/genética , Linhagem Celular , Humanos , Hibridização in Situ Fluorescente/métodos , Imagem Molecular/métodos , Saccharomycetales/metabolismo
8.
J Cell Biol ; 216(8): 2355-2371, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28637749

RESUMO

Telomerase can generate a novel telomere at DNA double-strand breaks (DSBs), an event called de novo telomere addition. How this activity is suppressed remains unclear. Combining single-molecule imaging and deep sequencing, we show that the budding yeast telomerase RNA (TLC1 RNA) is spatially segregated to the nucleolus and excluded from sites of DNA repair in a cell cycle-dependent manner. Although TLC1 RNA accumulates in the nucleoplasm in G1/S, Pif1 activity promotes TLC1 RNA localization in the nucleolus in G2/M. In the presence of DSBs, TLC1 RNA remains nucleolar in most G2/M cells but accumulates in the nucleoplasm and colocalizes with DSBs in rad52Δ cells, leading to de novo telomere additions. Nucleoplasmic accumulation of TLC1 RNA depends on Cdc13 localization at DSBs and on the SUMO ligase Siz1, which is required for de novo telomere addition in rad52Δ cells. This study reveals novel roles for Pif1, Rad52, and Siz1-dependent sumoylation in the spatial exclusion of telomerase from sites of DNA repair.


Assuntos
Ciclo Celular , Nucléolo Celular/enzimologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Fúngico/metabolismo , RNA Fúngico/metabolismo , RNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Telomerase/metabolismo , Telômero/metabolismo , Transporte Ativo do Núcleo Celular , Bleomicina/toxicidade , Ciclo Celular/efeitos dos fármacos , Nucléolo Celular/efeitos dos fármacos , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/efeitos dos fármacos , DNA Fúngico/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA/genética , RNA Fúngico/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Imagem Individual de Molécula , Sumoilação , Telomerase/genética , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Methods ; 114: 46-53, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27474163

RESUMO

In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs.


Assuntos
Imagem Molecular/métodos , RNA Fúngico/genética , RNA não Traduzido/genética , RNA/genética , Saccharomyces cerevisiae/genética , Telomerase/genética , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
10.
PLoS Genet ; 12(8): e1006268, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27564449

RESUMO

SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, nse3-1, are defective in Smc5/6 localization to telomeres and have slightly shorter telomeres. Nse3 interacts physically and genetically with two Rap1-binding factors, Rif2 and Sir4. Reduction in telomere-associated Smc5/6 leads to defects in telomere clustering, dispersion of the silencing factor, Sir4, and a loss in transcriptional repression for sub-telomeric genes and non-coding telomeric repeat-containing RNA (TERRA). SIR4 recovery at telomeres is reduced in cells lacking Smc5/6 functionality and vice versa. However, nse3-1/ sir4 Δ double mutants show additive defects for telomere shortening and TPE indicating the contribution of Smc5/6 to telomere homeostasis is only in partial overlap with SIR factor silencing. These findings support a role for Smc5/6 in telomere maintenance that is separate from its canonical role(s) in HR-mediated events during replication and telomere elongation.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Telômero/genética , Transcrição Gênica , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sumoilação/genética , Proteínas de Ligação a Telômeros/genética , Coesinas
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