RESUMO
Transcription-replication conflict is a major cause of replication stress that arises when replication forks collide with the transcription machinery. Replication fork stalling at sites of transcription compromises chromosome replication fidelity and can induce DNA damage with potentially deleterious consequences for genome stability and organismal health. The block to DNA replication by the transcription machinery is complex and can involve stalled or elongating RNA polymerases, promoter-bound transcription factor complexes, or DNA topology constraints. In addition, studies over the past two decades have identified co-transcriptional R-loops as a major source for impairment of DNA replication forks at active genes. However, how R-loops impede DNA replication at the molecular level is incompletely understood. Current evidence suggests that RNA:DNA hybrids, DNA secondary structures, stalled RNA polymerases, and condensed chromatin states associated with R-loops contribute to the of fork progression. Moreover, since both R-loops and replication forks are intrinsically asymmetric structures, the outcome of R-loop-replisome collisions is influenced by collision orientation. Collectively, the data suggest that the impact of R-loops on DNA replication is highly dependent on their specific structural composition. Here, we will summarize our current understanding of the molecular basis for R-loop-induced replication fork progression defects.
Assuntos
Replicação do DNA , Estruturas R-Loop , Humanos , DNA/química , Cromossomos , RNA Polimerases Dirigidas por DNA/genética , Instabilidade GenômicaRESUMO
To study the direct effects of R-loops on DNA replication and other DNA-templated processes in vitro, R-loop-containing DNA templates need to be prepared efficiently and to near homogeneity. Here, we describe a simple transcription-based approach to form R-loops on plasmid DNA templates in vitro. We detail steps to transcribe a DNA sequence element with a high propensity to form co-transcriptional R-loops using T7 RNA polymerase. We describe nucleolytic digestion of free RNA, deproteinization, and repurification of R-loop-containing templates via gel filtration. For complete details on the use and execution of this protocol, please refer to Kumar et al.1.
Assuntos
Estruturas R-Loop , Transcrição Gênica , RNA , Plasmídeos/genética , DNA/genéticaRESUMO
Sumoylation is emerging as a posttranslation modification important for regulating chromosome duplication and stability. The origin recognition complex (ORC) that directs DNA replication initiation by loading the MCM replicative helicase onto origins is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are unclear. Here we report the effects of hypersumoylation and hyposumoylation of yeast ORC on ORC activity and origin function using multiple approaches. ORC hypersumoylation preferentially reduced the function of a subset of early origins, while Orc2 hyposumoylation had an opposing effect. Mechanistically, ORC hypersumoylation reduced MCM loading in vitro and diminished MCM chromatin association in vivo. Either hypersumoylation or hyposumoylation of ORC resulted in genome instability and the dependence of yeast on other genome maintenance factors, providing evidence that appropriate ORC sumoylation levels are important for cell fitness. Thus, yeast ORC sumoylation status must be properly controlled to achieve optimal origin function across the genome and genome stability.
RESUMO
G-quadruplexes (G4s) are non-canonical nucleic acid structures that form in G-rich regions of the genome and threaten genome stability by interfering with DNA replication. However, the underlying mechanisms are poorly understood. We have recently found that G4s can stall eukaryotic replication forks by blocking the progression of replicative DNA helicase, CMG. In this paper, we detail the methodology of DNA unwinding assays to investigate the impact of G4s on CMG progression. The method details the purification of recombinantly expressed CMG from the budding yeast, Saccharomyces cerevisiae, purification of synthetic oligonucleotides, and covers various aspects of DNA substrate preparation, reaction setup and result interpretation. The use of synthetic oligonucleotides provides the advantage of allowing to control the formation of G4 structures in DNA substrates. The methods discussed here can be adapted for the study of other DNA helicases and provide a general template for the assembly of DNA substrates with distinct G4 structures.
Assuntos
Quadruplex G , DNA/química , DNA Helicases/química , Replicação do DNA , Oligonucleotídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3' end (3' ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.
Assuntos
DNA , Proteínas de Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Conformação Proteica , Proteína de Replicação C/química , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Formation of a properly sized and patterned embryo during gastrulation requires a well-coordinated interplay between cell proliferation, lineage specification and tissue morphogenesis. Following transient physical or pharmacological manipulations of embryo size, pre-gastrulation mouse embryos show remarkable plasticity to recover and resume normal development. However, it remains unclear how mechanisms driving lineage specification and morphogenesis respond to defects in cell proliferation during and after gastrulation. Null mutations in DNA replication or cell-cycle-related genes frequently lead to cell-cycle arrest and reduced cell proliferation, resulting in developmental arrest before the onset of gastrulation; such early lethality precludes studies aiming to determine the impact of cell proliferation on lineage specification and morphogenesis during gastrulation. From an unbiased ENU mutagenesis screen, we discovered a mouse mutant, tiny siren (tyrn), that carries a hypomorphic mutation producing an aspartate to tyrosine (D939Y) substitution in Pold1, the catalytic subunit of DNA polymerase δ. Impaired cell proliferation in the tyrn mutant leaves anterior-posterior patterning unperturbed during gastrulation but results in reduced embryo size and severe morphogenetic defects. Our analyses show that the successful execution of morphogenetic events during gastrulation requires that lineage specification and the ordered production of differentiated cell types occur in concordance with embryonic growth.
Assuntos
DNA Polimerase III , Gastrulação , Animais , DNA Polimerase III/genética , Embrião de Mamíferos , Gastrulação/genética , Camundongos , Morfogênese/genética , MutaçãoRESUMO
Fundamental to our understanding of chromosome duplication is the idea that replication origins function both as sites where MCM helicases are loaded during the G1 phase and where synthesis begins in S phase. However, the temporal delay between phases exposes the replisome assembly pathway to potential disruption prior to replication. Using multicolor, single-molecule imaging, we systematically study the consequences of encounters between actively transcribing RNA polymerases (RNAPs) and replication initiation intermediates in the context of chromatin. We demonstrate that RNAP can push multiple licensed MCM helicases over long distances with nucleosomes ejected or displaced. Unexpectedly, we observe that MCM helicase loading intermediates also can be repositioned by RNAP and continue origin licensing after encounters with RNAP, providing a web of alternative origin specification pathways. Taken together, our observations reveal a surprising mobility in origin-licensing factors that confers resistance to the complex challenges posed by diverse obstacles encountered on chromosomes.
Assuntos
Replicação do DNA , Origem de Replicação , Proteínas de Ciclo Celular/metabolismo , Cromatina , DNA Helicases/metabolismo , Replicação do DNA/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Origem de Replicação/genéticaRESUMO
Single-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATRMec1. However, the basis for 9-1-1's recruitment to 5' junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-replication factor C (RFC), in complex with 9-1-1 and a 5' junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1's recruitment to 5' junctions and reveal new principles of sliding clamp loading.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Trifosfato de Adenosina , Proteínas de Ciclo Celular , DNA/química , Replicação do DNA , Peptídeos e Proteínas de Sinalização Intracelular , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cdc6, a subunit of the pre-replicative complex (pre-RC), contains multiple regulatory cyclin-dependent kinase (Cdk1) consensus sites, SP or TP motifs. In Saccharomyces cerevisiae, Cdk1 phosphorylates Cdc6-T7 to recruit Cks1, the Cdk1 phospho-adaptor in S phase, for subsequent multisite phosphorylation and protein degradation. Cdc6 accumulates in mitosis and is tightly bound by Clb2 through N-terminal phosphorylation in order to prevent premature origin licensing and degradation. It has been extensively studied how Cdc6 phosphorylation is regulated by the cyclin-Cdk1 complex. However, a detailed mechanism on how Cdc6 phosphorylation is reversed by phosphatases has not been elucidated. Here, we show that PP2ACdc55 dephosphorylates Cdc6 N-terminal sites to release Clb2. Cdc14 dephosphorylates the C-terminal phospho-degron, leading to Cdc6 stabilization in mitosis. In addition, Cdk1 inhibitor Sic1 releases Clb2·Cdk1·Cks1 from Cdc6 to load Mcm2-7 on the chromatin upon mitotic exit. Thus, pre-RC assembly and origin licensing are promoted by phosphatases through the attenuation of distinct Cdk1-dependent Cdc6 inhibitory mechanisms.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Replicação do DNA/fisiologia , Proteína Fosfatase 2/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Mitose , Fosforilação , Saccharomyces cerevisiaeRESUMO
R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.
Assuntos
DNA/química , Quadruplex G , Hibridização de Ácido Nucleico , Estruturas R-Loop , RNA/química , Eletroforese em Gel de Ágar , Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Replicação do DNA/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA Fúngico/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiaeRESUMO
DNA polymerase epsilon (Pol ε) is required for genome duplication and tumor suppression. It supports both replisome assembly and leading strand synthesis; however, the underlying mechanisms remain to be elucidated. Here we report that a conserved domain within the Pol ε catalytic core influences both of these replication steps in budding yeast. Modeling cancer-associated mutations in this domain reveals its unexpected effect on incorporating Pol ε into the four-member pre-loading complex during replisome assembly. In addition, genetic and biochemical data suggest that the examined domain supports Pol ε catalytic activity and symmetric movement of replication forks. Contrary to previously characterized Pol ε cancer variants, the examined mutants cause genome hyper-rearrangement rather than hyper-mutation. Our work thus suggests a role of the Pol ε catalytic core in replisome formation, a reliance of Pol ε strand synthesis on a unique domain, and a potential tumor-suppressive effect of Pol ε in curbing genome re-arrangements.
Assuntos
DNA Polimerase II/fisiologia , Replicação do DNA , Regulação da Expressão Gênica , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Ciclo Celular , Proteínas de Ciclo Celular/genética , Estruturas Cromossômicas/genética , DNA Polimerase II/química , Doxiciclina/farmacologia , Genoma Humano , Humanos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Domínios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism of Rad53-dependent fork stabilization is not known. Using reconstituted budding yeast replisomes, we show that mutational inactivation of the leading strand DNA polymerase, Pol ε, dNTP depletion, and chemical inhibition of DNA polymerases cause excessive DNA unwinding by the replicative DNA helicase, CMG, demonstrating that budding yeast replisomes lack intrinsic mechanisms that control helicase-polymerase coupling at the fork. Importantly, we find that the Rad53 kinase restricts excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork stabilization by the replication checkpoint.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , DNA Fúngico/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , DNA Helicases/genética , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Primers do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Plasmídeos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , DNA Polimerase III/metabolismo , DNA Polimerase II/metabolismo , Replicação do DNA/fisiologia , DNA Fúngico/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , DNA Polimerase II/genética , DNA Polimerase III/genética , DNA Fúngico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2-5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2-5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC-Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/genética , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , Replicação do DNA , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Hidrólise , Microscopia Eletrônica , Modelos Moleculares , Complexo de Reconhecimento de Origem/metabolismo , Conformação Proteica , Domínios Proteicos , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication.
Assuntos
Cromatina/genética , Replicação do DNA , DNA Fúngico/genética , Nucleossomos/genética , Origem de Replicação , Saccharomyces cerevisiae/genética , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA Fúngico/biossíntese , Genótipo , Humanos , Nucleossomos/metabolismo , Fenótipo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de TempoRESUMO
The eukaryotic replicative DNA helicase, Mcm2-7, is loaded in inactive form as a double hexameric complex around double-stranded DNA. To ensure that replication origins fire no more than once per S phase, activation of the Mcm2-7 helicase is temporally separated from Mcm2-7 loading in the cell cycle. This 2-step mechanism requires that inactive Mcm2-7 complexes be maintained for variable periods of time in a topologically bound state on chromatin, which may create a steric obstacle to other DNA transactions. We have recently found in the budding yeast, Saccharomyces cerevisiae, that Mcm2-7 double hexamers can respond to collisions with transcription complexes by sliding along the DNA template. Importantly, Mcm2-7 double hexamers remain functional after displacement along DNA and support replication initiation from sites distal to the origin. These results reveal a novel mechanism to specify eukaryotic replication origin sites and to maintain replication origin competence without the need for Mcm2-7 reloading.
Assuntos
Eucariotos/genética , Origem de Replicação , Animais , DNA/biossíntese , DNA/genética , DNA/metabolismo , Ativação Enzimática , Humanos , Proteínas de Manutenção de Minicromossomo/metabolismo , Transcrição GênicaRESUMO
Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic replication origins are not irrevocably defined by selection of the helicase loading site, but can shift in position after helicase loading. Using purified proteins we show that DNA translocases, including RNA polymerase, can push budding yeast Mcm2-7 double hexamers along DNA. Displaced Mcm2-7 double hexamers support DNA replication initiation distal to the loading site in vitro. Similarly, in yeast cells that are defective for transcription termination, collisions with RNA polymerase induce a redistribution of Mcm2-7 complexes along the chromosomes, resulting in a corresponding shift in DNA replication initiation sites. These results reveal a eukaryotic origin specification mechanism that departs from the classical replicon model, helping eukaryotic cells to negotiate transcription-replication conflict.
Assuntos
Proteínas de Manutenção de Minicromossomo/metabolismo , Origem de Replicação , Saccharomyces cerevisiae/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA Fúngico/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The separation of DNA replication origin licensing and activation in the cell cycle is essential for genome stability across generations in eukaryotic cells. Pre-replicative complexes (pre-RCs) license origins by loading Mcm2-7 complexes in inactive form around DNA. During origin firing in S phase, replisomes assemble around the activated Mcm2-7 DNA helicase. Budding yeast pre-RCs have previously been reconstituted in vitro with purified proteins. Here, we show that reconstituted pre-RCs support replication of plasmid DNA in yeast cell extracts in a reaction that exhibits hallmarks of cellular replication initiation. Plasmid replication in vitro results in the generation of covalently closed circular daughter molecules, indicating that the system recapitulates the initiation, elongation, and termination stages of DNA replication. Unexpectedly, yeast origin DNA is not strictly required for DNA replication in vitro, as heterologous DNA sequences could support replication of plasmid molecules. Our findings support the notion that epigenetic mechanisms are important for determining replication origin sites in budding yeast, highlighting mechanistic principles of replication origin specification that are common among eukaryotes.
Assuntos
Replicação do DNA/fisiologia , Epigênese Genética/fisiologia , Proteínas de Manutenção de Minicromossomo/metabolismo , Modelos Biológicos , Plasmídeos/fisiologia , Origem de Replicação/genética , Saccharomycetales/fisiologia , Pegada de DNA , Primers do DNA/genética , Desoxirribonuclease I/genética , Escherichia coli , Plasmídeos/genética , Saccharomycetales/genéticaRESUMO
The regulated loading of the Mcm2-7 DNA helicase (comprising six related subunits, Mcm2 to Mcm7) into pre-replicative complexes at multiple replication origins ensures precise once per cell cycle replication in eukaryotic cells. The origin recognition complex (ORC), Cdc6 and Cdt1 load Mcm2-7 into a double hexamer bound around duplex DNA in an ATP-dependent reaction, but the molecular mechanism of this origin 'licensing' is still poorly understood. Here we show that both Mcm2-7 hexamers in Saccharomyces cerevisiae are recruited to origins by an essential, conserved carboxy-terminal domain of Mcm3 that interacts with and stimulates the ATPase activity of ORC-Cdc6. ATP hydrolysis can promote Mcm2-7 loading, but can also promote Mcm2-7 release if components are missing or if ORC has been inactivated by cyclin-dependent kinase phosphorylation. Our work provides new insights into how origins are licensed and reveals a novel ATPase-dependent mechanism contributing to precise once per cell cycle replication.