RESUMEN
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.
Asunto(s)
Cromatina/genética , Replicación del ADN , ADN de Hongos/genética , Nucleosomas/genética , Origen de Réplica , Saccharomyces cerevisiae/genética , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismo , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ADN de Hongos/biosíntesis , Genotipo , Humanos , Nucleosomas/metabolismo , Fenotipo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de TiempoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/metabolismo , ADN Polimerasa III/metabolismo , ADN Polimerasa II/metabolismo , Replicación del ADN/fisiología , ADN de Hongos/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2/genética , ADN Polimerasa II/genética , ADN Polimerasa III/genética , ADN de Hongos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
Replicación del ADN/fisiología , Epigénesis Genética/fisiología , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Modelos Biológicos , Plásmidos/fisiología , Origen de Réplica/genética , Saccharomycetales/fisiología , Huella de ADN , Cartilla de ADN/genética , Desoxirribonucleasa I/genética , Escherichia coli , Plásmidos/genética , Saccharomycetales/genéticaRESUMEN
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.
Asunto(s)
G-Cuádruplex , ADN/química , ADN Helicasas/química , Replicación del ADN , Oligonucleótidos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
ADN Polimerasa III , Gastrulación , Animales , ADN Polimerasa III/genética , Embrión de Mamíferos , Gastrulación/genética , Ratones , Morfogénesis/genética , MutaciónRESUMEN
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.
Asunto(s)
ADN , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfato/metabolismo , ADN/metabolismo , Replicación del ADN , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Conformación Proteica , Proteína de Replicación C/química , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , ADN de Hongos/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2/genética , ADN Helicasas/genética , ADN Polimerasa II/genética , ADN Polimerasa II/metabolismo , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Cartilla de ADN , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Plásmidos , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
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.