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1.
Sci Adv ; 10(23): eadn7191, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38848361

RESUMO

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson's disease (PD). Stabilization of PINK1 at the translocase of outer membrane (TOM) complex of damaged mitochondria is critical for its activation. The mechanism of how PINK1 is activated in the TOM complex is unclear. Here, we report that co-expression of human PINK1 and all seven TOM subunits in Saccharomyces cerevisiae is sufficient for PINK1 activation. We use this reconstitution system to systematically assess the role of each TOM subunit toward PINK1 activation. We unambiguously demonstrate that the TOM20 and TOM70 receptor subunits are required for optimal PINK1 activation and map their sites of interaction with PINK1 using AlphaFold structural modeling and mutagenesis. We also demonstrate an essential role of the pore-containing subunit TOM40 and its structurally associated subunits TOM7 and TOM22 for PINK1 activation. These findings will aid in the development of small-molecule activators of PINK1 as a therapeutic strategy for PD.


Assuntos
Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Quinases , Saccharomyces cerevisiae , Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Humanos , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Mitocôndrias/metabolismo , Ligação Proteica , Ativação Enzimática , Modelos Moleculares , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética
2.
EMBO Rep ; 24(11): e57677, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37781960

RESUMO

DONSON is one of 13 genes mutated in a form of primordial microcephalic dwarfism known as Meier-Gorlin syndrome. The other 12 encode components of the CDC45-MCM-GINS helicase, around which the eukaryotic replisome forms, or are factors required for helicase assembly during DNA replication initiation. A role for DONSON in CDC45-MCM-GINS assembly was unanticipated, since DNA replication initiation can be reconstituted in vitro with purified proteins from budding yeast, which lacks DONSON. Using mouse embryonic stem cells as a model for the mammalian helicase, we show that DONSON binds directly but transiently to CDC45-MCM-GINS during S-phase and is essential for chromosome duplication. Rapid depletion of DONSON leads to the disappearance of the CDC45-MCM-GINS helicase from S-phase cells and our data indicate that DONSON is dispensable for loading of the MCM2-7 helicase core onto chromatin during G1-phase, but instead is essential for CDC45-MCM-GINS assembly during S-phase. These data identify DONSON as a missing link in our understanding of mammalian chromosome duplication and provide a molecular explanation for why mutations in human DONSON are associated with Meier-Gorlin syndrome.


Assuntos
Proteínas de Ciclo Celular , Duplicação Cromossômica , Camundongos , Animais , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Replicação do DNA , Proteínas de Manutenção de Minicromossomo/metabolismo , Mamíferos/metabolismo
3.
Science ; 381(6664): eadi4932, 2023 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-37590372

RESUMO

Assembly of the CMG (CDC-45-MCM-2-7-GINS) helicase is the key regulated step during eukaryotic DNA replication initiation. Until now, it was unclear whether metazoa require additional factors that are not present in yeast. In this work, we show that Caenorhabditis elegans DNSN-1, the ortholog of human DONSON, functions during helicase assembly in a complex with MUS-101/TOPBP1. DNSN-1 is required to recruit the GINS complex to chromatin, and a cryo-electron microscopy structure indicates that DNSN-1 positions GINS on the MCM-2-7 helicase motor (comprising the six MCM-2 to MCM-7 proteins), by direct binding of DNSN-1 to GINS and MCM-3, using interfaces that we show are important for initiation and essential for viability. These findings identify DNSN-1 as a missing link in our understanding of DNA replication initiation, suggesting that initiation defects underlie the human disease syndrome that results from DONSON mutations.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Replicação do DNA , Proteínas de Manutenção de Minicromossomo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Domínios Proteicos
4.
Elife ; 112022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35920641

RESUMO

The p97/Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 are essential to unfold ubiquitylated proteins in many areas of eukaryotic cell biology. In yeast, Cdc48-Ufd1-Npl4 is controlled by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here, we show that mammalian p97-UFD1-NPL4 is governed by a complex interplay between additional p97 cofactors and the number of conjugated ubiquitins. Using reconstituted assays for the disassembly of ubiquitylated CMG (Cdc45-MCM-GINS) helicase by human p97-UFD1-NPL4, we show that the unfoldase has a high ubiquitin threshold for substrate unfolding, which can be reduced by the UBX proteins UBXN7, FAF1, or FAF2. Our data indicate that the UBX proteins function by binding to p97-UFD1-NPL4 and stabilising productive interactions between UFD1-NPL4 and K48-linked chains of at least five ubiquitins. Stimulation by UBXN7 is dependent upon known ubiquitin-binding motifs, whereas FAF1 and FAF2 use a previously uncharacterised coiled-coil domain to reduce the ubiquitin threshold of p97-UFD1-NPL4. We show that deleting the Ubnx7 and Faf1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Nucleares , Ubiquitinas , Proteína com Valosina , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Ubiquitinas/metabolismo , Proteína com Valosina/metabolismo
5.
EMBO J ; 41(5): e109783, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35102600

RESUMO

Nucleosomes are disrupted transiently during eukaryotic transcription, yet the displaced histones must be retained and redeposited onto DNA, to preserve nucleosome density and associated histone modifications. Here, we show that the essential Spt5 processivity factor of RNA polymerase II (Pol II) plays a direct role in this process in budding yeast. Functional orthologues of eukaryotic Spt5 are present in archaea and bacteria, reflecting its universal role in RNA polymerase processivity. However, eukaryotic Spt5 is unique in having an acidic amino terminal tail (Spt5N) that is sandwiched between the downstream nucleosome and the upstream DNA that emerges from Pol II. We show that Spt5N contains a histone-binding motif that is required for viability in yeast cells and prevents loss of nucleosomal histones within actively transcribed regions. These findings indicate that eukaryotic Spt5 combines two essential activities, which together couple processive transcription to the efficient capture and re-deposition of nucleosomal histones.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Histonas/genética , RNA Polimerase II/genética , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/genética , Nucleossomos/genética , Ligação Proteica/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
6.
Nature ; 600(7890): 743-747, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34700328

RESUMO

Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase1-3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia2 in budding yeast1, CUL2LRR1 in metazoa4-7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA8-10. However, it is unknown how SCFDia2 and CUL2LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome-E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR-MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity.


Assuntos
Replicação do DNA , Eucariotos , Microscopia Crioeletrônica , DNA/metabolismo , DNA Helicases/metabolismo , Eucariotos/genética , Humanos , Ubiquitina-Proteína Ligases/metabolismo
7.
EMBO J ; 40(17): e108053, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34269473

RESUMO

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin-RING ubiquitin ligase CUL-2LRR-1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC-48 "unfoldase". Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome-associated TIMELESS-TIPIN complex is required for CUL-2LRR-1 recruitment and efficient CMG helicase ubiquitylation. Aided by TIMELESS-TIPIN, CUL-2LRR-1 directs a suite of ubiquitylation enzymes to ubiquitylate the MCM-7 subunit of CMG. Subsequently, the UBXN-3 adaptor protein directly stimulates the disassembly of ubiquitylated CMG by CDC-48_UFD-1_NPL-4. We show that UBXN-3 is important in vivo for replisome disassembly in the absence of TIMELESS-TIPIN. Correspondingly, co-depletion of UBXN-3 and TIMELESS causes profound synthetic lethality. Since the human orthologue of UBXN-3, FAF1, is a candidate tumour suppressor, these findings suggest that manipulation of CMG disassembly might be applicable to future strategies for treating human cancer.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Proteínas Culina/genética , Proteínas Culina/metabolismo , Mutações Sintéticas Letais
8.
Biochem J ; 478(14): 2825-2842, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34195792

RESUMO

Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.


Assuntos
Proteínas Culina/metabolismo , DNA Helicases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Linhagem Celular , Clonagem Molecular/métodos , Proteínas Culina/genética , DNA Helicases/genética , Humanos , Immunoblotting , Lisina/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Sf9 , Spodoptera , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
9.
Biochem J ; 478(13): 2465-2479, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198324

RESUMO

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed. SARS-CoV-2 encodes 16 non-structural proteins (nsp) that possess different enzymatic activities with important roles in viral genome replication, transcription and host immune evasion. One key aspect of host immune evasion is performed by the uridine-directed endoribonuclease activity of nsp15. Here we describe the expression and purification of nsp15 recombinant protein. We have developed biochemical assays to follow its activity, and we have found evidence for allosteric behaviour. We screened a custom chemical library of over 5000 compounds to identify nsp15 endoribonuclease inhibitors, and we identified and validated NSC95397 as an inhibitor of nsp15 endoribonuclease in vitro. Although NSC95397 did not inhibit SARS-CoV-2 growth in VERO E6 cells, further studies will be required to determine the effect of nsp15 inhibition on host immune evasion.


Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Endorribonucleases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Animais , Chlorocebus aethiops , Endorribonucleases/isolamento & purificação , Endorribonucleases/metabolismo , Ensaios Enzimáticos , Fluorescência , Ensaios de Triagem em Larga Escala , Técnicas In Vitro , Cinética , Naftoquinonas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/química , Soluções , Células Vero , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo
10.
Biochem J ; 478(13): 2517-2531, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198325

RESUMO

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.


Assuntos
Antivirais/química , Antivirais/farmacologia , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Compostos de Anilina/farmacologia , Animais , Benzamidas/farmacologia , Chlorocebus aethiops , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/isolamento & purificação , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Sinergismo Farmacológico , Ensaios Enzimáticos , Flavinas/farmacologia , Transferência Ressonante de Energia de Fluorescência , Furanos/farmacologia , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Naftalenos/farmacologia , Fenantrenos/farmacologia , Quinonas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Bibliotecas de Moléculas Pequenas/química , Células Vero , Replicação Viral/efeitos dos fármacos
11.
Biochem J ; 478(13): 2445-2464, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198326

RESUMO

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.


Assuntos
Antivirais/química , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exorribonucleases/antagonistas & inibidores , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Animais , Ácido Aurintricarboxílico/farmacologia , Chlorocebus aethiops , Ensaios Enzimáticos , Exorribonucleases/metabolismo , Fluorescência , Ensaios de Triagem em Larga Escala , Patulina/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Células Vero , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo
12.
Biochem J ; 478(13): 2499-2515, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198327

RESUMO

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.


Assuntos
Antivirais/química , Antivirais/farmacologia , Proteases 3C de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Azóis/farmacologia , Chlorocebus aethiops , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/isolamento & purificação , Proteases 3C de Coronavírus/metabolismo , Ensaios Enzimáticos , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Isoindóis , Leupeptinas/farmacologia , Compostos Organosselênicos/farmacologia , Peptidomiméticos , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Células Vero , Proteínas não Estruturais Virais/metabolismo
13.
Biochem J ; 478(13): 2481-2497, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34198328

RESUMO

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2'-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Exorribonucleases/antagonistas & inibidores , Metiltransferases/antagonistas & inibidores , Capuzes de RNA/metabolismo , SARS-CoV-2/enzimologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Antivirais/química , Clorobenzenos/farmacologia , Chlorocebus aethiops , Ensaios Enzimáticos , Exorribonucleases/genética , Exorribonucleases/isolamento & purificação , Exorribonucleases/metabolismo , Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Indazóis/farmacologia , Indenos/farmacologia , Indóis/farmacologia , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Nitrilas/farmacologia , Fenotiazinas/farmacologia , Purinas/farmacologia , Reprodutibilidade dos Testes , SARS-CoV-2/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato , Trifluperidol/farmacologia , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/isolamento & purificação , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/isolamento & purificação , Proteínas Virais Reguladoras e Acessórias/metabolismo
14.
EMBO Rep ; 22(3): e52164, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33590678

RESUMO

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2LRR1 is required for ubiquitylation of the CMG-MCM7 subunit during S-phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis-regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.


Assuntos
DNA Helicases , Replicação do DNA , Animais , Ciclo Celular/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Camundongos , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Ubiquitinação , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo
15.
Elife ; 92020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32804080

RESUMO

The eukaryotic replisome assembles around the CMG helicase, which stably associates with DNA replication forks throughout elongation. When replication terminates, CMG is ubiquitylated on its Mcm7 subunit and disassembled by the Cdc48/p97 ATPase. Until now, the regulation that restricts CMG ubiquitylation to termination was unknown, as was the mechanism of disassembly. By reconstituting these processes with purified budding yeast proteins, we show that ubiquitylation is tightly repressed throughout elongation by the Y-shaped DNA structure of replication forks. Termination removes the repressive DNA structure, whereupon long K48-linked ubiquitin chains are conjugated to CMG-Mcm7, dependent on multiple replisome components that bind to the ubiquitin ligase SCFDia2. This mechanism pushes CMG beyond a '5-ubiquitin threshold' that is inherent to Cdc48, which specifically unfolds ubiquitylated Mcm7 and thereby disassembles CMG. These findings explain the exquisite regulation of CMG disassembly and provide a general model for the disassembly of ubiquitylated protein complexes by Cdc48.


Assuntos
DNA Helicases , Replicação do DNA , DNA , Ubiquitina , DNA/química , DNA/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Escherichia coli , Humanos , Conformação de Ácido Nucleico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Proteína com Valosina/química , Proteína com Valosina/metabolismo
16.
Cell Rep ; 28(11): 2777-2783.e4, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31509741

RESUMO

Disassembly of the replisome is the final step of chromosome duplication in eukaryotes. In budding yeast and metazoa, cullin ubiquitin ligases are required to ubiquitylate the Cdc45-MCM-GINS (CMG) helicase that lies at the heart of the replisome, leading to a disassembly reaction that is dependent upon the ATPase known as Cdc48 or p97. Here, we describe the reconstitution of replisome disassembly, using a purified complex of the budding yeast replisome in association with the cullin ligase SCFDia2. Upon addition of E1 and E2 enzymes, together with ubiquitin and ATP, the CMG helicase is ubiquitylated on its Mcm7 subunit. Subsequent addition of Cdc48, together with its cofactors Ufd1-Npl4, drives efficient disassembly of ubiquitylated CMG, thereby recapitulating the steps of replisome disassembly that are observed in vivo. Our findings define the minimal requirements for disassembly of the eukaryotic replisome and provide a model system for studying the disassembly of protein complexes by Cdc48-Ufd1-Npl4.


Assuntos
DNA Helicases/metabolismo , Proteínas F-Box/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Ubiquitinação/genética , Proteína com Valosina/metabolismo , DNA Helicases/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas In Vitro , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína com Valosina/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
17.
Elife ; 82019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31545170

RESUMO

The faithful segregation of eukaryotic chromosomes in mitosis requires that the genome be duplicated completely prior to anaphase. However, cells with large genomes sometimes fail to complete replication during interphase and instead enter mitosis with regions of incompletely replicated DNA. These regions are processed in early mitosis via a process known as mitotic DNA repair synthesis (MiDAS), but little is known about how cells switch from conventional DNA replication to MiDAS. Using the early embryo of the nematode Caenorhabditis elegans as a model system, we show that the TRAIP ubiquitin ligase drives replisome disassembly in response to incomplete DNA replication, thereby providing access to replication forks for other factors. Moreover, TRAIP is essential for MiDAS in human cells, and is important in both systems to prevent mitotic segregation errors. Our data indicate that TRAIP is a master regulator of the processing of incomplete DNA replication during mitosis in metazoa.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Reparo do DNA , Replicação do DNA , Mitose , Ubiquitina-Proteína Ligases/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Linhagem Celular , Deleção de Genes , Humanos , Ubiquitina-Proteína Ligases/genética
18.
Mol Cell ; 73(5): 915-929.e6, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849395

RESUMO

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Dano ao DNA , Replicação do DNA , DNA/biossíntese , Rearranjo Gênico , Mitose , Proteínas Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , DNA/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo , DNA Polimerase teta
19.
Mol Cell ; 74(2): 231-244.e9, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30850330

RESUMO

The convergence of two DNA replication forks creates unique problems during DNA replication termination. In E. coli and SV40, the release of torsional strain by type II topoisomerases is critical for converging replisomes to complete DNA synthesis, but the pathways that mediate fork convergence in eukaryotes are unknown. We studied the convergence of reconstituted yeast replication forks that include all core replisome components and both type I and type II topoisomerases. We found that most converging forks stall at a very late stage, indicating a role for additional factors. We showed that the Pif1 and Rrm3 DNA helicases promote efficient fork convergence and completion of DNA synthesis, even in the absence of type II topoisomerase. Furthermore, Rrm3 and Pif1 are also important for termination of plasmid DNA replication in vivo. These findings identify a eukaryotic pathway for DNA replication termination that is distinct from previously characterized prokaryotic mechanisms.


Assuntos
DNA Helicases/genética , Replicação do DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Topoisomerases/genética , Escherichia coli/genética , Eucariotos/genética , Instabilidade Genômica , Plasmídeos/genética
20.
Mol Cell ; 72(1): 140-151.e3, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30244834

RESUMO

Although essential for epigenetic inheritance, the transfer of parental histone (H3-H4)2 tetramers that contain epigenetic modifications to replicating DNA strands is poorly understood. Here, we show that the Mcm2-Ctf4-Polα axis facilitates the transfer of parental (H3-H4)2 tetramers to lagging-strand DNA at replication forks. Mutating the conserved histone-binding domain of the Mcm2 subunit of the CMG (Cdc45-MCM-GINS) DNA helicase, which translocates along the leading-strand template, results in a marked enrichment of parental (H3-H4)2 on leading strand, due to the impairment of the transfer of parental (H3-H4)2 to lagging strands. Similar effects are observed in Ctf4 and Polα primase mutants that disrupt the connection of the CMG helicase to Polα that resides on lagging-strand template. Our results support a model whereby parental (H3-H4)2 complexes displaced from nucleosomes by DNA unwinding at replication forks are transferred by the CMG-Ctf4-Polα complex to lagging-strand DNA for nucleosome assembly at the original location.


Assuntos
DNA Polimerase III/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , Epigênese Genética , Histonas/genética , Complexos Multiproteicos/genética , Nucleossomos/genética , Ligação Proteica , Saccharomyces cerevisiae/genética
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