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1.
Nucleic Acids Res ; 46(3): 1227-1239, 2018 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-29059325

RESUMO

The yeast RNA/DNA helicase Sen1, Senataxin in human, preserves the integrity of replication forks encountering transcription by removing RNA-DNA hybrids. Here we show that, in sen1 mutants, when a replication fork clashes head-on with transcription is arrested and, as a consequence, the progression of the sister fork moving in the opposite direction within the same replicon is also impaired. Therefore, sister forks remain coupled when one of the two forks is arrested by transcription, a fate different from that experienced by forks encountering Double Strand Breaks. We also show that dormant origins of replication are activated to ensure DNA synthesis in the proximity to the forks arrested by transcription. Dormant origin firing is not inhibited by the replication checkpoint, rather dormant origins are fired if they cannot be timely inactivated by passive replication. In sen1 mutants, the Mre11 and Mrc1-Ctf4 complexes protect the forks arrested by transcription from processing mediated by the Exo1 nuclease. Thus, a harmless head-on replication-transcription clash resolution requires the fine-tuning of origin firing and coordination among Sen1, Exo1, Mre11 and Mrc1-Ctf4 complexes.


Assuntos
DNA Helicases/genética , Replicação do DNA , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Regulação Fúngica da Expressão Gênica , RNA Helicases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Mutação , Ligação Proteica , RNA Helicases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Nat Commun ; 15(1): 2890, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38570537

RESUMO

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Saccharomyces cerevisiae , Humanos , Ciclo Celular , Recombinação Homóloga , Divisão Celular , Endonucleases/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , DNA , Reparo do DNA , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
Life Sci Alliance ; 6(9)2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37468166

RESUMO

Fun30 is the prototype of the Fun30-SMARCAD1-ETL subfamily of nucleosome remodelers involved in DNA repair and gene silencing. These proteins appear to act as single-subunit nucleosome remodelers, but their molecular mechanisms are, at this point, poorly understood. Using multiple sequence alignment and structure prediction, we identify an evolutionarily conserved domain that is modeled to contain a SAM-like fold with one long, protruding helix, which we term SAM-key. Deletion of the SAM-key within budding yeast Fun30 leads to a defect in DNA repair and gene silencing similar to that of the fun30Δ mutant. In vitro, Fun30 protein lacking the SAM-key is able to bind nucleosomes but is deficient in DNA-stimulated ATPase activity and nucleosome sliding and eviction. A structural model based on AlphaFold2 prediction and verified by crosslinking-MS indicates an interaction of the long SAM-key helix with protrusion I, a subdomain located between the two ATPase lobes that is critical for control of enzymatic activity. Mutation of the interaction interface phenocopies the domain deletion with a lack of DNA-stimulated ATPase activation and a nucleosome-remodeling defect, thereby confirming a role of the SAM-key helix in regulating ATPase activity. Our data thereby demonstrate a central role of the SAM-key domain in mediating the activation of Fun30 catalytic activity, thus highlighting the importance of allosteric activation for this class of enzymes.


Assuntos
Nucleossomos , Proteínas de Saccharomyces cerevisiae , Nucleossomos/genética , Nucleossomos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo
5.
Front Genet ; 12: 821543, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35096025

RESUMO

DNA double strand breaks (DSBs) are repaired in eukaryotes by one of several cellular mechanisms. The decision-making process controlling DSB repair takes place at the step of DNA end resection, the nucleolytic processing of DNA ends, which generates single-stranded DNA overhangs. Dependent on the length of the overhang, a corresponding DSB repair mechanism is engaged. Interestingly, nucleosomes-the fundamental unit of chromatin-influence the activity of resection nucleases and nucleosome remodelers have emerged as key regulators of DSB repair. Nucleosome remodelers share a common enzymatic mechanism, but for global genome organization specific remodelers have been shown to exert distinct activities. Specifically, different remodelers have been found to slide and evict, position or edit nucleosomes. It is an open question whether the same remodelers exert the same function also in the context of DSBs. Here, we will review recent advances in our understanding of nucleosome remodelers at DSBs: to what extent nucleosome sliding, eviction, positioning and editing can be observed at DSBs and how these activities affect the DSB repair decision.

6.
Elife ; 92020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32352375

RESUMO

Cell cycle tags allow to restrict target protein expression to specific cell cycle phases. Here, we present an advanced toolbox of cell cycle tag constructs in budding yeast with defined and compatible peak expression that allow comparison of protein functionality at different cell cycle phases. We apply this technology to the question of how and when Mus81-Mms4 and Yen1 nucleases act on DNA replication or recombination structures. Restriction of Mus81-Mms4 to M phase but not S phase allows a wildtype response to various forms of replication perturbation and DNA damage in S phase, suggesting it acts as a post-replicative resolvase. Moreover, we use cell cycle tags to reinstall cell cycle control to a deregulated version of Yen1, showing that its premature activation interferes with the response to perturbed replication. Curbing resolvase activity and establishing a hierarchy of resolution mechanisms are therefore the principal reasons underlying resolvase cell cycle regulation.


Assuntos
Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Mitose/fisiologia , Dano ao DNA/fisiologia , Endonucleases Flap/metabolismo , Fase S/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Cell Rep ; 17(2): 556-569, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27705801

RESUMO

Temporal separation of DNA replication initiation into licensing and firing phases ensures the precise duplication of the genome during each cell cycle. Cyclin-dependent kinase (CDK) is known to generate this separation by activating firing factors and at the same time inhibiting licensing factors but may not be sufficient to ensure robust separation at transitions between both phases. Here, we show that a temporal gap separates the inactivation of firing factors from the re-activation of licensing factors during mitosis in budding yeast. We find that gap size critically depends on phosphorylation-dependent degradation of the firing factor Sld2 mediated by CDK, DDK, Mck1, and Cdc5 kinases and the ubiquitin-ligases Dma1/2. Stable mutants of Sld2 minimize the gap and cause increased genome instability in an origin-dependent manner when combined with deregulation of other replication regulators or checkpoint mechanisms. Robust separation of licensing and firing phases therefore appears indispensable to safeguard genome stability.


Assuntos
Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/metabolismo , Genoma Fúngico , Instabilidade Genômica/genética , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Front Genet ; 6: 166, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25972894

RESUMO

DNA replication and transcription are vital cellular processes during which the genetic information is copied into complementary DNA and RNA molecules. Highly complex machineries required for DNA and RNA synthesis compete for the same DNA template, therefore being on a collision course. Unscheduled replication-transcription clashes alter the gene transcription program and generate replication stress, reducing fork speed. Molecular pathways and mechanisms that minimize the conflict between replication and transcription have been extensively characterized in prokaryotic cells and recently identified also in eukaryotes. A pathological outcome of replication-transcription collisions is the formation of stable RNA:DNA hybrids in molecular structures called R-loops. Growing evidence suggests that R-loop accumulation promotes both genetic and epigenetic instability, thus severely affecting genome functionality. In the present review, we summarize the current knowledge related to replication and transcription conflicts in eukaryotes, their consequences on genome stability and the pathways involved in their resolution. These findings are relevant to clarify the molecular basis of cancer and neurodegenerative diseases.

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