Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 33
Filtrar
1.
Annu Rev Biochem ; 90: 77-106, 2021 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-33784179

RESUMO

The faithful and timely copying of DNA by molecular machines known as replisomes depends on a disparate suite of enzymes and scaffolding factors working together in a highly orchestrated manner. Large, dynamic protein-nucleic acid assemblies that selectively morph between distinct conformations and compositional states underpin this critical cellular process. In this article, we discuss recent progress outlining the physical basis of replisome construction and progression in eukaryotes.


Assuntos
Replicação do DNA , DNA/biossíntese , Eucariotos/genética , Complexo de Reconhecimento de Origem/metabolismo , Animais , DNA/química , DNA Polimerase III/química , DNA Polimerase III/metabolismo , Humanos , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/genética , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo
2.
Nature ; 519(7543): 321-6, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25762138

RESUMO

Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. Here we describe the crystal structure of Drosophila ORC at 3.5 Å resolution, showing that the 270 kilodalton initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1-5 subunits sits atop a layer of AAA+ (ATPases associated with a variety of cellular activities) folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident. These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2-7 (MCM2-7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.


Assuntos
Drosophila melanogaster/química , Células Eucarióticas/química , Complexo de Reconhecimento de Origem/química , Animais , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Replicação do DNA , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/metabolismo , Modelos Biológicos , Modelos Moleculares , Complexo de Reconhecimento de Origem/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rotação
3.
Proc Natl Acad Sci U S A ; 115(26): E5906-E5915, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29899147

RESUMO

In eukaryotes, the heterohexameric origin recognition complex (ORC) coordinates replication onset by facilitating the recruitment and loading of the minichromosome maintenance 2-7 (Mcm2-7) replicative helicase onto DNA to license origins. Drosophila ORC can adopt an autoinhibited configuration that is predicted to prevent Mcm2-7 loading; how the complex is activated and whether other ORC homologs can assume this state are not known. Using chemical cross-linking and mass spectrometry, biochemical assays, and electron microscopy (EM), we show that the autoinhibited state of Drosophila ORC is populated in solution, and that human ORC can also adopt this form. ATP binding to ORC supports a transition from the autoinhibited state to an active configuration, enabling the nucleotide-dependent association of ORC with both DNA and Cdc6. An unstructured N-terminal region adjacent to the conserved ATPase domain of Orc1 is shown to be required for high-affinity ORC-DNA interactions, but not for activation. ORC optimally binds DNA duplexes longer than the predicted footprint of the ORC ATPases associated with a variety of cellular activities (AAA+) and winged-helix (WH) folds; cryo-EM analysis of Drosophila ORC bound to DNA and Cdc6 indicates that ORC contacts DNA outside of its central core region, bending the DNA away from its central DNA-binding channel. Our findings indicate that ORC autoinhibition may be common to metazoans and that ORC-Cdc6 remodels origin DNA before Mcm2-7 recruitment and loading.


Assuntos
DNA/química , Proteínas de Manutenção de Minicromossomo/química , Complexo de Reconhecimento de Origem/química , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Complexo de Reconhecimento de Origem/metabolismo
4.
Crit Rev Biochem Mol Biol ; 52(2): 107-144, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28094588

RESUMO

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.


Assuntos
Replicação do DNA , Células Eucarióticas/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Ativação Enzimática , Humanos , Proteínas de Manutenção de Minicromossomo/análise , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexo de Reconhecimento de Origem/análise , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação
5.
Mol Cell ; 41(5): 543-53, 2011 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-21362550

RESUMO

Eukaryotic cells license far more origins than are actually used for DNA replication, thereby generating a large number of dormant origins. Accumulating evidence suggests that such origins play a role in chromosome stability and tumor suppression, though the underlying mechanism is largely unknown. Here, we show that a loss of dormant origins results in an increased number of stalled replication forks, even in unchallenged S phase in primary mouse fibroblasts derived from embryos homozygous for the Mcm4(Chaos3) allele. We found that this allele reduces the stability of the MCM2-7 complex, but confers normal helicase activity in vitro. Despite the activation of multiple fork recovery pathways, replication intermediates in these cells persist into M phase, increasing the number of abnormal anaphase cells with lagging chromosomes and/or acentric fragments. These findings suggest that dormant origins constitute a major pathway for stalled fork recovery, contributing to faithful chromosome segregation and tumor suppression.


Assuntos
Neoplasias/patologia , Fase S , Alelos , Anáfase , Animais , Ciclo Celular , Divisão Celular , Instabilidade Cromossômica , Segregação de Cromossomos , Citocinese , DNA Helicases/metabolismo , Replicação do DNA , Fibroblastos/citologia , Camundongos , Rad51 Recombinase/metabolismo , Recombinação Genética
6.
Mol Cell ; 37(2): 247-58, 2010 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-20122406

RESUMO

MCM2-7 proteins provide essential helicase functions in eukaryotes at chromosomal DNA replication forks. During the G1 phase of the cell cycle, they remain loaded on DNA but are inactive. We have used recombinant methods to show that the Drosophila MCM2-7 helicase is activated in complex with Cdc45 and the four GINS proteins (CMG complex). Biochemical activities of the MCM AAA+ motor are altered and enhanced through such associations: ATP hydrolysis rates are elevated by two orders of magnitude, helicase activity is robust on circular templates, and affinity for DNA substrates is improved. The GINS proteins contribute to DNA substrate affinity and bind specifically to the MCM4 subunit. All pairwise associations among GINS, MCMs, and Cdc45 were detected, but tight association takes place only in the CMG. The onset of DNA replication and unwinding may thus occur through allosteric changes in MCM2-7 affected by the association of these ancillary factors.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , DNA/metabolismo , Replicação do DNA , Proteínas de Drosophila/química , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ativação Enzimática , Modelos Biológicos , Dados de Sequência Molecular , Alinhamento de Sequência
7.
Proc Natl Acad Sci U S A ; 112(3): E249-58, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25561522

RESUMO

DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2-7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2-7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2-7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel.


Assuntos
DNA Helicases/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , DNA/metabolismo , Corantes Fluorescentes , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
8.
Proc Natl Acad Sci U S A ; 111(40): E4234-43, 2014 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-25249635

RESUMO

Lethal malignant brain tumors (lmbt) result from the loss of the conserved transcriptional repressor l(3)mbt, in Drosophila melanogaster. Similar mutations in the human homolog L3MBTL1 correlate with some cancers. The protein's C-terminal MBT repeats bind mono and dimethylated histones in vitro, which could influence recruitment of L3MBTL1 to its target sites. The L(3)mbt chromatin targeting mechanism, however, is controversial and several studies suggest insufficiency or a minor role for histone methylation in determining the site specificity for recruitment. We report that L(3)mbt colocalizes with core members of the Myb-MuvB/DREAM (MMB/DREAM) transcriptional regulatory complex genome-wide, and that L(3)mbt-mediated repression requires this complex in salivary glands and larval brains. Loss of l(3)mbt or of MMB components through mutation cause similar spurious expression of genes, including the transposon regulatory gene piwi, in terminally differentiated cells. The DNA-binding MMB core component Mip120 (Lin54) is required for L(3)mbt recruitment to chromosomes, whereas Mip130 (Lin9) (an MMB core protein) and E2f2 (an MMB transcriptional repressor) are not, but are essential for repression. Cytolocalization experiments suggest the presence of site-specific differential composition of MMB in polytene chromosomes where some loci were bound by a Myb-containing or alternatively, an E2f2 and L(3)mbt form of the complex.


Assuntos
Proteínas de Transporte/metabolismo , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Cromossomos de Insetos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/genética , Caspases/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cromatina/genética , Cromossomos de Insetos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-myb/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Salivares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Proc Natl Acad Sci U S A ; 109(33): 13163-70, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22853956

RESUMO

The replication of eukaryote chromosomes slows down when DNA is damaged and the proteins that work at the fork (the replisome) are known targets for the signaling pathways that mediate such responses critical for accurate genomic inheritance. However, the molecular mechanisms and details of how this response is mediated are poorly understood. In this report we show that the activity of replisome helicase, the Cdc45/MCM2-7/GINS (CMG) complex, can be inhibited by protein phosphorylation. Recombinant Drosophila melanogaster CMG can be stimulated by treatment with phosphatase whereas Chk2 but not Chk1 interferes with the helicase activity in vitro. The targets for Chk2 phosphorylation have been identified and reside in MCM subunits 3 and 4 and in the GINS protein Psf2. Interference requires a combination of modifications and we suggest that the formation of negative charges might create a surface on the helicase to allosterically affect its function. The treatment of developing fly embryos with ionizing radiation leads to hyperphosphorylation of Psf2 subunit in the active helicase complex. Taken together these data suggest that the direct modification of the CMG helicase by Chk2 is an important nexus for response to DNA damage.


Assuntos
DNA Helicases/antagonistas & inibidores , Replicação do DNA , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Quinase do Ponto de Checagem 2 , DNA Helicases/metabolismo , Replicação do DNA/efeitos da radiação , Drosophila melanogaster/embriologia , Drosophila melanogaster/efeitos da radiação , Embrião não Mamífero/enzimologia , Embrião não Mamífero/efeitos da radiação , Fosforilação/efeitos da radiação , Subunidades Proteicas/metabolismo , Radiação Ionizante
10.
Proc Natl Acad Sci U S A ; 109(30): 11999-2004, 2012 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-22778422

RESUMO

The heterohexameric minichromosome maintenance (MCM2-7) complex is an ATPase that serves as the central replicative helicase in eukaryotes. During initiation, the ring-shaped MCM2-7 particle is thought to open to facilitate loading onto DNA. The conformational state accessed during ring opening, the interplay between ATP binding and MCM2-7 architecture, and the use of these events in the regulation of DNA unwinding are poorly understood. To address these issues in isolation from the regulatory complexity of existing eukaryotic model systems, we investigated the structure/function relationships of a naturally minimized MCM2-7 complex from the microsporidian parasite Encephalitozoon cuniculi. Electron microscopy and small-angle X-ray scattering studies show that, in the absence of ATP, MCM2-7 spontaneously adopts a left-handed, open-ring structure. Nucleotide binding does not promote ring closure but does cause the particle to constrict in a two-step process that correlates with the filling of high- and low-affinity ATPase sites. Our findings support the idea that an open ring forms the default conformational state of the isolated MCM2-7 complex, and they provide a structural framework for understanding the multiphasic ATPase kinetics observed in different MCM2-7 systems.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Encephalitozoon cuniculi/enzimologia , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Conformação Proteica , Trifosfato de Adenosina/química , DNA Helicases/química , Proteínas de Ligação a DNA/química , Ativação Enzimática , Microscopia Eletrônica , Complexos Multiproteicos/química , Espalhamento a Baixo Ângulo
11.
PLoS Genet ; 7(10): e1002266, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22028662

RESUMO

The earliest stages of development in most metazoans are driven by maternally deposited proteins and mRNAs, with widespread transcriptional activation of the zygotic genome occurring hours after fertilization, at a period known as the maternal-to-zygotic transition (MZT). In Drosophila, the MZT is preceded by the transcription of a small number of genes that initiate sex determination, patterning, and other early developmental processes; and the zinc-finger protein Zelda (ZLD) plays a key role in their transcriptional activation. To better understand the mechanisms of ZLD activation and the range of its targets, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to map regions bound by ZLD before (mitotic cycle 8), during (mitotic cycle 13), and after (late mitotic cycle 14) the MZT. Although only a handful of genes are transcribed prior to mitotic cycle 10, we identified thousands of regions bound by ZLD in cycle 8 embryos, most of which remain bound through mitotic cycle 14. As expected, early ZLD-bound regions include the promoters and enhancers of genes transcribed at this early stage. However, we also observed ZLD bound at cycle 8 to the promoters of roughly a thousand genes whose first transcription does not occur until the MZT and to virtually all of the thousands of known and presumed enhancers bound at cycle 14 by transcription factors that regulate patterned gene activation during the MZT. The association between early ZLD binding and MZT activity is so strong that ZLD binding alone can be used to identify active promoters and regulatory sequences with high specificity and selectivity. This strong early association of ZLD with regions not active until the MZT suggests that ZLD is not only required for the earliest wave of transcription but also plays a major role in activating the genome at the MZT.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Zigoto/crescimento & desenvolvimento , Animais , Padronização Corporal/genética , Imunoprecipitação da Cromatina/métodos , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Genoma , Mitose , Proteínas Nucleares , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Processos de Determinação Sexual/genética , Fatores de Transcrição/genética , Transcrição Gênica , Zigoto/metabolismo
12.
Dev Biol ; 345(2): 248-55, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20599892

RESUMO

Maternally contributed mRNAs and proteins control the initial stages of development following fertilization. During this time, most of the zygotic genome remains transcriptionally silent. The initiation of widespread zygotic transcription is coordinated with the degradation of maternally provided mRNAs at the maternal-to-zygotic transition (MZT). While most of the genome is silenced prior to the MZT, a small subset of zygotic genes essential for the future development of the organism is transcribed. Previous work in our laboratory and others identified the TAGteam element, a set of related heptameric DNA-sequences in the promoters of many early-expressed Drosophila genes required to drive their unusually early transcription. To understand how this unique subset of genes is regulated, we identified a TAGteam-binding factor Grainyhead (Grh). We demonstrated that Grh and the previously characterized transcriptional activator Zelda (Zld) bind to different TAGteam sequences with varying affinities, and that Grh competes with Zld for TAGteam occupancy. Moreover, overexpression of Grh in the early embryo causes defects in cell division, phenocopying Zld depletion. Our findings indicate that during early embryonic development the precise timing of gene expression is regulated by both the sequence of the TAGteam elements in the promoter and the relative levels of the transcription factors Grh and Zld.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Embrião não Mamífero/metabolismo , Feminino , Proteínas Nucleares , Fatores de Transcrição/genética
13.
Elife ; 102021 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-34951585

RESUMO

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using Drosophila melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.


Assuntos
Fenômenos Bioquímicos , Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , Drosophila melanogaster/genética , Proteínas Intrinsicamente Desordenadas/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Drosophila melanogaster/fisiologia , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/metabolismo
14.
Elife ; 82019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31560342

RESUMO

The initiation of DNA replication in metazoans occurs at thousands of chromosomal sites known as origins. At each origin, the Origin Recognition Complex (ORC), Cdc6, and Cdt1 co-assemble to load the Mcm2-7 replicative helicase onto chromatin. Current replication models envisage a linear arrangement of isolated origins functioning autonomously; the extent of inter-origin organization and communication is unknown. Here, we report that the replication initiation machinery of D. melanogaster unexpectedly undergoes liquid-liquid phase separation (LLPS) upon binding DNA in vitro. We find that ORC, Cdc6, and Cdt1 contain intrinsically disordered regions (IDRs) that drive LLPS and constitute a new class of phase separating elements. Initiator IDRs are shown to regulate multiple functions, including chromosome recruitment, initiator-specific co-assembly, and Mcm2-7 loading. These data help explain how CDK activity controls replication initiation and suggest that replication programs are subject to higher-order levels of inter-origin organization.


Assuntos
Replicação do DNA , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Complexo de Reconhecimento de Origem/metabolismo , Animais , Cromatina/metabolismo , Multimerização Proteica
15.
Cell Rep ; 28(10): 2673-2688.e8, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484077

RESUMO

In the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Eucariotos/enzimologia , Complexos Multienzimáticos/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica , DNA/ultraestrutura , DNA Helicases/química , DNA Helicases/ultraestrutura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Hidrólise , Modelos Moleculares , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo
16.
Curr Biol ; 15(8): 755-9, 2005 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-15854909

RESUMO

The full complement of proteins required for the proper regulation of genome duplication are yet to be described. We employ a genetic DNA-replication model system based on developmental amplification of Drosophila eggshell (chorion) genes [1]. Hypomorphic mutations in essential DNA replication genes result in a distinct thin-eggshell phenotype owing to reduced amplification [2]. Here, we molecularly identify the gene, which we have named humpty dumpty (hd), corresponding to the thin-eggshell mutant fs(3)272-9 [3]. We confirm that hd is essential for DNA amplification in the ovary and show that it also is required for cell proliferation during development. Mosaic analysis of hd mutant cells during development and RNAi in Kc cells reveal that depletion of Hd protein results in severe defects in genomic replication and DNA damage. Most Hd protein is found in nuclear foci, and some may traverse the nuclear envelope. Consistent with a role in DNA replication, expression of Hd protein peaks during late G1 and S phase, and it responds to the E2F1/Dp transcription factor. Hd protein sequence is conserved from plants to humans, and published microarrays indicate that expression of its putative human ortholog also peaks at G1/S [4]. Our data suggest that hd defines a new gene family likely required for cell proliferation in all multicellular eukaryotes.


Assuntos
Proliferação de Células , Córion/citologia , Replicação do DNA/genética , Proteínas de Drosophila/genética , Drosophila/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Encéfalo/metabolismo , Células Cultivadas , Clonagem Molecular , Primers do DNA , Drosophila/fisiologia , Proteínas de Drosophila/metabolismo , Feminino , Citometria de Fluxo , Microscopia de Fluorescência , Dados de Sequência Molecular , Família Multigênica/genética , Ovário/metabolismo , Interferência de RNA , Alinhamento de Sequência , Análise de Sequência de DNA , Transgenes/genética
17.
Science ; 355(6327)2017 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-28209641

RESUMO

Cellular DNA replication factories depend on ring-shaped hexameric helicases to aid DNA synthesis by processively unzipping the parental DNA helix. Replicative helicases are loaded onto DNA by dedicated initiator, loader, and accessory proteins during the initiation of DNA replication in a tightly regulated, multistep process. We discuss here the molecular choreography of DNA replication initiation across the three domains of life, highlighting similarities and differences in the strategies used to deposit replicative helicases onto DNA and to melt the DNA helix in preparation for replisome assembly. Although initiators and loaders are phylogenetically related, the mechanisms they use for accomplishing similar tasks have diverged considerably and in an unpredictable manner.


Assuntos
Células/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , Archaea/enzimologia , Archaea/genética , Archaea/metabolismo , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Células/enzimologia , DNA Helicases/química , Eucariotos/enzimologia , Eucariotos/genética , Eucariotos/metabolismo , Sequências Hélice-Volta-Hélice , Filogenia , Domínios Proteicos
18.
Elife ; 3: e03273, 2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-25117490

RESUMO

The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3' single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/metabolismo , Complexos Multiproteicos/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , DNA/metabolismo , Replicação do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Eucarióticas/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo
19.
Cold Spring Harb Perspect Biol ; 5(11): a012807, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23881943

RESUMO

The eukaryotic replicative helicase, the minichromosome maintenance (MCM) complex, is composed of six distinct, but related, subunits MCM(2-7). The relationship between the sequences of the subunits indicates that they are derived from a common ancestor and indeed, present-day archaea possess a homohexameric MCM. Recent progress in the biochemical and structural studies of both eukaryal and archaeal MCM complexes are beginning to shed light on the mechanisms of action of this key component of the replisome.


Assuntos
Archaea/metabolismo , DNA Helicases/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Proteínas Arqueais/metabolismo , Sistema Livre de Células , Drosophila melanogaster , Methanobacteriaceae/metabolismo , Modelos Moleculares , Plasmídeos/metabolismo , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Xenopus
20.
Elife ; 2: e00882, 2013 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-24137536

RESUMO

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2-7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions. DOI:http://dx.doi.org/10.7554/eLife.00882.001.


Assuntos
Microtia Congênita/genética , Transtornos do Crescimento/genética , Micrognatismo/genética , Mutação , Complexo de Reconhecimento de Origem/genética , Patela/anormalidades , Animais , Drosophila , Humanos , Microscopia Eletrônica , Complexo de Reconhecimento de Origem/ultraestrutura
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA