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1.
Science ; 376(6597): 1087-1094, 2022 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-35653469

RESUMO

Structural maintenance of chromosomes (SMC) protein complexes structure genomes by extruding DNA loops, but the molecular mechanism that underlies their activity has remained unknown. We show that the active condensin complex entraps the bases of a DNA loop transiently in two separate chambers. Single-molecule imaging and cryo-electron microscopy suggest a putative power-stroke movement at the first chamber that feeds DNA into the SMC-kleisin ring upon adenosine triphosphate binding, whereas the second chamber holds on upstream of the same DNA double helix. Unlocking the strict separation of "motor" and "anchor" chambers turns condensin from a one-sided into a bidirectional DNA loop extruder. We conclude that the orientation of two topologically bound DNA segments during the SMC reaction cycle determines the directionality of DNA loop extrusion.


Assuntos
Adenosina Trifosfatases , Proteínas de Ligação a DNA , DNA , Complexos Multiproteicos , Adenosina Trifosfatases/química , Microscopia Crioeletrônica , DNA/química , Proteínas de Ligação a DNA/química , Complexos Multiproteicos/química , Conformação de Ácido Nucleico , Imagem Individual de Molécula
2.
Cells ; 10(3)2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806417

RESUMO

Most Cyclin-dependent kinases (Cdks) are redundant for normal cell division. Here we tested whether these redundancies are maintained during cell cycle recovery after a DNA damage-induced arrest in G1. Using non-transformed RPE-1 cells, we find that while Cdk4 and Cdk6 act redundantly during normal S-phase entry, they both become essential for S-phase entry after DNA damage in G1. We show that this is due to a greater overall dependency for Cdk4/6 activity, rather than to independent functions of either kinase. In addition, we show that inactivation of pocket proteins is sufficient to overcome the inhibitory effects of complete Cdk4/6 inhibition in otherwise unperturbed cells, but that this cannot revert the effects of Cdk4/6 inhibition in DNA damaged cultures. Indeed, we could confirm that, in addition to inactivation of pocket proteins, Cdh1-dependent anaphase-promoting complex/cyclosome (APC/CCdh1) activity needs to be inhibited to promote S-phase entry in damaged cultures. Collectively, our data indicate that DNA damage in G1 creates a unique situation where high levels of Cdk4/6 activity are required to inactivate pocket proteins and APC/CCdh1 to promote the transition from G1 to S phase.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Dano ao DNA/genética , Fase G1/fisiologia , Humanos , Transfecção
3.
Nature ; 579(7799): 438-442, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132705

RESUMO

Condensin, a key component of the structure maintenance of chromosome (SMC) protein complexes, has recently been shown to be a motor that extrudes loops of DNA1. It remains unclear, however, how condensin complexes work together to collectively package DNA into chromosomes. Here we use time-lapse single-molecule visualization to study mutual interactions between two DNA-loop-extruding yeast condensins. We find that these motor proteins, which, individually, extrude DNA in one direction only are able to dynamically change each other's DNA loop sizes, even when far apart. When they are in close proximity, condensin complexes are able to traverse each other and form a loop structure, which we term a Z-loop-three double-stranded DNA helices aligned in parallel with one condensin at each edge. Z-loops can fill gaps left by single loops and can form symmetric dimer motors that pull in DNA from both sides. These findings indicate that condensin may achieve chromosomal compaction using a variety of looping structures.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Proteínas Motores Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Conformação Proteica , Saccharomyces cerevisiae , Adenosina Trifosfatases/química , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/química , Cromossomos/metabolismo , Proteínas de Ligação a DNA/química , Proteínas Motores Moleculares/química , Complexos Multiproteicos/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Imagem com Lapso de Tempo
4.
Mol Cell ; 74(6): 1175-1188.e9, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31226277

RESUMO

The condensin protein complex plays a key role in the structural organization of genomes. How the ATPase activity of its SMC subunits drives large-scale changes in chromosome topology has remained unknown. Here we reconstruct, at near-atomic resolution, the sequence of events that take place during the condensin ATPase cycle. We show that ATP binding induces a conformational switch in the Smc4 head domain that releases its hitherto undescribed interaction with the Ycs4 HEAT-repeat subunit and promotes its engagement with the Smc2 head into an asymmetric heterodimer. SMC head dimerization subsequently enables nucleotide binding at the second active site and disengages the Brn1 kleisin subunit from the Smc2 coiled coil to open the condensin ring. These large-scale transitions in the condensin architecture lay out a mechanistic path for its ability to extrude DNA helices into large loop structures.


Assuntos
Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Proteínas de Transporte/química , Chaetomium/genética , Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , DNA/química , Complexos Multiproteicos/química , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Chaetomium/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Cromossomos/ultraestrutura , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Células HeLa , Humanos , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
5.
Curr Biol ; 28(21): R1266-R1281, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30399354

RESUMO

Protein complexes built of structural maintenance of chromosomes (SMC) and kleisin subunits, including cohesin, condensin and the Smc5/6 complex, are master organizers of genome architecture in all kingdoms of life. How these large ring-shaped molecular machines use the energy of ATP hydrolysis to change the topology of chromatin fibers has remained a central unresolved question of chromosome biology. A currently emerging concept suggests that the common principle that underlies the essential functions of SMC protein complexes in the control of gene expression, chromosome segregation or DNA damage repair is their ability to expand DNA into large loop structures. Here, we review the current knowledge about the biochemical and structural properties of SMC protein complexes that might enable them to extrude DNA loops and compare their action to other motor proteins and nucleic acid translocases. We evaluate the currently predominant models of active loop extrusion and propose a detailed version of a 'scrunching' model, which reconciles much of the available mechanistic data and provides an elegant explanation for how SMC protein complexes fulfill an array of seemingly diverse tasks during the organization of genomes.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cromossomos/fisiologia , Complexos Multiproteicos/fisiologia , Proteínas de Transporte/genética , Proteínas Cromossômicas não Histona/genética , Humanos
6.
Science ; 360(6384): 102-105, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29472443

RESUMO

It has been hypothesized that SMC protein complexes such as condensin and cohesin spatially organize chromosomes by extruding DNA into large loops. We directly visualized the formation and processive extension of DNA loops by yeast condensin in real time. Our findings constitute unambiguous evidence for loop extrusion. We observed that a single condensin complex is able to extrude tens of kilobase pairs of DNA at a force-dependent speed of up to 1500 base pairs per second, using the energy of adenosine triphosphate hydrolysis. Condensin-induced loop extrusion was strictly asymmetric, which demonstrates that condensin anchors onto DNA and reels it in from only one side. Active DNA loop extrusion by SMC complexes may provide the universal unifying principle for genome organization.


Assuntos
Adenosina Trifosfatases/química , Proteínas de Ligação a DNA/química , DNA/química , Complexos Multiproteicos/química , Conformação de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/química , Imagem Individual de Molécula/métodos , Trifosfato de Adenosina/química , Hidrólise , Fatores de Tempo
7.
J Cell Sci ; 128(4): 607-20, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25609713

RESUMO

Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cycle. Reversal of a DNA-damage-induced checkpoint not only requires the repair of these lesions, but a cell must also prevent permanent exit from the cell cycle and actively terminate checkpoint signalling to allow cell cycle progression to resume. It is becoming increasingly clear that despite the shared mechanisms of DNA damage detection throughout the cell cycle, the checkpoint and its reversal are precisely tuned to each cell cycle phase. Furthermore, recent findings challenge the dogmatic view that complete repair is a precondition for cell cycle resumption. In this Commentary, we highlight cell-cycle-dependent differences in checkpoint signalling and recovery after a DNA DSB, and summarise the molecular mechanisms that underlie the reversal of DNA damage checkpoints, before discussing when and how cell fate decisions after a DSB are made.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas de Ciclo Celular/genética , Divisão Celular , DNA/genética , Humanos , Transdução de Sinais/genética
8.
PLoS Genet ; 10(10): e1004594, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25340510

RESUMO

We recently reported that centrosomal protein 164 (CEP164) regulates both cilia and the DNA damage response in the autosomal recessive polycystic kidney disease nephronophthisis. Here we examine the functional role of CEP164 in nephronophthisis-related ciliopathies and concomitant fibrosis. Live cell imaging of RPE-FUCCI (fluorescent, ubiquitination-based cell cycle indicator) cells after siRNA knockdown of CEP164 revealed an overall quicker cell cycle than control cells, although early S-phase was significantly longer. Follow-up FACS experiments with renal IMCD3 cells confirm that Cep164 siRNA knockdown promotes cells to accumulate in S-phase. We demonstrate that this effect can be rescued by human wild-type CEP164, but not disease-associated mutants. siRNA of CEP164 revealed a proliferation defect over time, as measured by CyQuant assays. The discrepancy between accelerated cell cycle and inhibited overall proliferation could be explained by induction of apoptosis and epithelial-to-mesenchymal transition. Reduction of CEP164 levels induces apoptosis in immunofluorescence, FACS and RT-QPCR experiments. Furthermore, knockdown of Cep164 or overexpression of dominant negative mutant allele CEP164 Q525X induces epithelial-to-mesenchymal transition, and concomitant upregulation of genes associated with fibrosis. Zebrafish injected with cep164 morpholinos likewise manifest developmental abnormalities, impaired DNA damage signaling, apoptosis and a pro-fibrotic response in vivo. This study reveals a novel role for CEP164 in the pathogenesis of nephronophthisis, in which mutations cause ciliary defects coupled with DNA damage induced replicative stress, cell death, and epithelial-to-mesenchymal transition, and suggests that these events drive the characteristic fibrosis observed in nephronophthisis kidneys.


Assuntos
Cílios/genética , Fibrose/genética , Doenças Renais Císticas/genética , Proteínas dos Microtúbulos/genética , Animais , Apoptose/genética , Ciclo Celular/genética , Cílios/patologia , Dano ao DNA/genética , Transição Epitelial-Mesenquimal , Fibrose/patologia , Técnicas de Silenciamento de Genes , Humanos , Doenças Renais Císticas/patologia , Proteínas dos Microtúbulos/biossíntese , RNA Interferente Pequeno , Transdução de Sinais , Peixe-Zebra
9.
Mol Cell Biol ; 34(22): 4177-85, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25202122

RESUMO

In response to genotoxic stress, DNA damage checkpoints maintain the integrity of the genome by delaying cell cycle progression to allow for DNA repair. Here we show that the degradation of the basic helix-loop-helix (bHLH) transcription factor DEC1, a critical regulator of cell fate and circadian rhythms, controls the DNA damage response. During unperturbed cell cycles, DEC1 is a highly unstable protein that is targeted for proteasome-dependent degradation by the SCF(ßTrCP) ubiquitin ligase in cooperation with CK1. Upon DNA damage, DEC1 is rapidly induced in an ATM/ATR-dependent manner. DEC1 induction results from protein stabilization via a mechanism that requires the USP17 ubiquitin protease. USP17 binds and deubiquitylates DEC1, markedly extending its half-life. Subsequently, during checkpoint recovery, DEC1 proteolysis is reestablished through ßTrCP-dependent ubiquitylation. Expression of a degradation-resistant DEC1 mutant prevents checkpoint recovery by inhibiting the downregulation of p53. These results indicate that the regulated degradation of DEC1 is a key factor controlling the DNA damage response.


Assuntos
Dano ao DNA , Endopeptidases/metabolismo , Proteólise , Proteínas Supressoras de Tumor/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Ciclo Celular , Linhagem Celular , Regulação para Baixo , Humanos , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitinação
10.
Mol Cell ; 55(1): 59-72, 2014 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-24910099

RESUMO

DNA damage can result in a transient cell-cycle arrest or lead to permanent cell-cycle withdrawal. Here we show that the decision to irreversibly withdraw from the cell cycle is made within a few hours following damage in G2 cells. This permanent arrest is dependent on induction of p53 and p21, resulting in the nuclear retention of Cyclin B1. This rapid response is followed by the activation of the APC/C(Cdh1) (the anaphase-promoting complex/cyclosome and its coactivator Cdh1) several hours later. Inhibition of APC/C(Cdh1) activity fails to prevent cell-cycle withdrawal, whereas preventing nuclear retention of Cyclin B1 does allow cells to remain in cycle. Importantly, transient induction of p53 in G2 cells is sufficient to induce senescence. Taken together, these results indicate that a rapid and transient pulse of p53 in G2 can drive nuclear retention of Cyclin B1 as the first irreversible step in the onset of senescence.


Assuntos
Senescência Celular/genética , Dano ao DNA , Fase G2 , Proteína Supressora de Tumor p53/fisiologia , Transporte Ativo do Núcleo Celular , Pontos de Checagem do Ciclo Celular , Diferenciação Celular , Ciclina B1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Proteína Supressora de Tumor p53/metabolismo
11.
Proc Natl Acad Sci U S A ; 111(20): 7313-8, 2014 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-24711418

RESUMO

The basic machinery that detects DNA damage is the same throughout the cell cycle. Here, we show, in contrast, that reversal of DNA damage responses (DDRs) and recovery are fundamentally different in G1 and G2 phases of the cell cycle. We find that distinct phosphatases are required to counteract the checkpoint response in G1 vs. G2. Whereas WT p53-induced phosphatase 1 (Wip1) promotes recovery in G2-arrested cells by antagonizing p53, it is dispensable for recovery from a G1 arrest. Instead, we identify phosphoprotein phosphatase 4 catalytic subunit (PP4) to be specifically required for cell cycle restart after DNA damage in G1. PP4 dephosphorylates Krüppel-associated box domain-associated protein 1-S473 to repress p53-dependent transcriptional activation of p21 when the DDR is silenced. Taken together, our results show that PP4 and Wip1 are differentially required to counteract the p53-dependent cell cycle arrest in G1 and G2, by antagonizing early or late p53-mediated responses, respectively.


Assuntos
Regulação Neoplásica da Expressão Gênica , Fosfoproteínas Fosfatases/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Ciclo Celular , Quinase do Ponto de Checagem 2/metabolismo , Ciclina B1/metabolismo , DNA/genética , Dano ao DNA , Fibroblastos/metabolismo , Fase G1/efeitos da radiação , Fase G2/efeitos da radiação , Humanos , Proteínas Luminescentes/metabolismo , Mutação , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteína Fosfatase 2C , Estrutura Terciária de Proteína , Epitélio Pigmentado da Retina/citologia , Telomerase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
J Cell Biol ; 201(4): 511-21, 2013 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-23649806

RESUMO

The DNA damage response (DDR) pathway and its core component tumor suppressor p53 block cell cycle progression after genotoxic stress and represent an intrinsic barrier preventing cancer development. The serine/threonine phosphatase PPM1D/Wip1 inactivates p53 and promotes termination of the DDR pathway. Wip1 has been suggested to act as an oncogene in a subset of tumors that retain wild-type p53. In this paper, we have identified novel gain-of-function mutations in exon 6 of PPM1D that result in expression of C-terminally truncated Wip1. Remarkably, mutations in PPM1D are present not only in the tumors but also in other tissues of breast and colorectal cancer patients, indicating that they arise early in development or affect the germline. We show that mutations in PPM1D affect the DDR pathway and propose that they could predispose to cancer.


Assuntos
Fase G1 , Regulação Neoplásica da Expressão Gênica , Mutação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/fisiologia , Proteína Supressora de Tumor p53/genética , Ciclo Celular , Linhagem Celular Tumoral , Dano ao DNA , Predisposição Genética para Doença , Células HeLa , Humanos , Células MCF-7 , Neoplasias/metabolismo , Proteína Fosfatase 2C
13.
J Virol ; 84(4): 2134-49, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20007278

RESUMO

Coronaviruses induce in infected cells the formation of double-membrane vesicles (DMVs) in which the replication-transcription complexes (RTCs) are anchored. To study the dynamics of these coronavirus replicative structures, we generated recombinant murine hepatitis coronaviruses that express tagged versions of the nonstructural protein nsp2. We demonstrated by using immunofluorescence assays and electron microscopy that this protein is recruited to the DMV-anchored RTCs, for which its C terminus is essential. Live-cell imaging of infected cells demonstrated that small nsp2-positive structures move through the cytoplasm in a microtubule-dependent manner. In contrast, large fluorescent structures are rather immobile. Microtubule-mediated transport of DMVs, however, is not required for efficient replication. Biochemical analyses indicated that the nsp2 protein is associated with the cytoplasmic side of the DMVs. Yet, no recovery of fluorescence was observed when (part of) the nsp2-positive foci were bleached. This result was confirmed by the observation that preexisting RTCs did not exchange fluorescence after fusion of cells expressing either a green or a red fluorescent nsp2. Apparently, nsp2, once recruited to the RTCs, is not exchanged with nsp2 present in the cytoplasm or at other DMVs. Our data show a remarkable resemblance to results obtained recently by others with hepatitis C virus. The observations point to intriguing and as yet unrecognized similarities between the RTC dynamics of different plus-strand RNA viruses.


Assuntos
Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/fisiologia , Animais , Sequência de Bases , Gatos , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Vesículas Citoplasmáticas/virologia , Primers do DNA/genética , DNA Viral/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Substâncias Macromoleculares , Camundongos , Microscopia Eletrônica de Transmissão , Vírus da Hepatite Murina/patogenicidade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia
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