RESUMO
The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What impact this has on the MCM double hexamer is unknown. Here, we used cryoelectron microscopy (cryo-EM) on proteins isolated from replicating Xenopus egg extracts to identify a double CMG complex bridged by a DONSON dimer. We find that tethering elements mediating complex formation are essential for replication. DONSON reconfigures the MCM motors in the double CMG, and primordial dwarfism patients' mutations disrupting DONSON dimerization affect GINS and MCM engagement in human cells and DNA synthesis in Xenopus egg extracts.
Assuntos
Proteínas de Ciclo Celular , DNA Helicases , Proteínas Nucleares , Animais , Humanos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , DNA/genética , DNA/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Ativação EnzimáticaRESUMO
Pol epsilon is a tetrameric assembly that plays distinct roles during eukaryotic chromosome replication. It catalyses leading strand DNA synthesis; yet this function is dispensable for viability. Its non-catalytic domains instead play an essential role in the assembly of the active replicative helicase and origin activation, while non-essential histone-fold subunits serve a critical function in parental histone redeposition onto newly synthesised DNA. Furthermore, Pol epsilon plays a structural role in linking the RFC-Ctf18 clamp loader to the replisome, supporting processive DNA synthesis, DNA damage response signalling as well as sister chromatid cohesion. In this minireview, we discuss recent biochemical and structural work that begins to explain various aspects of eukaryotic chromosome replication, with a focus on the multiple roles of Pol epsilon in this process.
Assuntos
Proteínas de Saccharomyces cerevisiae , Cromossomos/metabolismo , DNA/genética , DNA Polimerase II/metabolismo , Replicação do DNA , Histonas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The exosome is a large molecular machine involved in RNA degradation and processing. Here we address how the trimeric Rrp4 cap enhances the activity of the archaeal enzyme complex. Using methyl-TROSY NMR methods we identified a 50-Å long RNA binding path on each Rrp4 protomer. We show that the Rrp4 cap can thus simultaneously recruit three substrates, one of which is degraded in the core while the others are positioned for subsequent degradation rounds. The local interaction energy between the substrate and the Rrp4-exosome increases from the periphery of the complex toward the active sites. Notably, the intrinsic interaction strength between the cap and the substrate is weakened as soon as substrates enter the catalytic barrel, which provides a means to reduce friction during substrate movements toward the active sites. Our data thus reveal a sophisticated exosome-substrate interaction mechanism that enables efficient RNA degradation.
Assuntos
Proteínas Arqueais/metabolismo , Exossomos/metabolismo , RNA Arqueal/metabolismo , Sulfolobus solfataricus/metabolismo , Proteínas Arqueais/química , Exossomos/química , Ressonância Magnética Nuclear Biomolecular , RNA Arqueal/química , Sulfolobus solfataricus/químicaRESUMO
The exosome plays an important role in RNA degradation and processing. In archaea, three Rrp41:Rrp42 heterodimers assemble into a barrel like structure that contains a narrow RNA entrance pore and a lumen that contains three active sites. Here, we demonstrate that this quaternary structure of the exosome is important for efficient RNA degradation. We find that the entrance pore of the barrel is required for nM substrate affinity. This strong interaction is crucial for processive substrate degradation and prevents premature release of the RNA from the enzyme. Using methyl TROSY NMR techniques, we establish that the 3' end of the substrate remains highly flexible inside the lumen. As a result, the RNA jumps between the three active sites that all equally participate in substrate degradation. The RNA jumping rate is, however, much faster than the cleavage rate, indicating that not all active site:substrate encounters result in catalysis. Enzymatic turnover therefore benefits from the confinement of the active sites and substrate in the lumen, which ensures that the RNA is at all times bound to one of the active sites. The evolution of the exosome into a hexameric complex and the optimization of its catalytic efficiency were thus likely co-occurring events.
Assuntos
Proteínas Arqueais/química , Complexo Multienzimático de Ribonucleases do Exossomo/química , Exossomos/química , RNA Arqueal/química , Proteínas de Ligação a RNA/química , Sulfolobus solfataricus/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Biocatálise , Domínio Catalítico , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/enzimologia , Expressão Gênica , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Estabilidade de RNA , RNA Arqueal/genética , RNA Arqueal/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Sulfolobus solfataricus/química , Sulfolobus solfataricus/genéticaRESUMO
Here, we present a large-scale FLAG immunoprecipitation protocol to isolate large protein complexes driving DNA replication at replicating chromatin assembled in Xenopus laevis egg extract. We describe how to prepare demembranated sperm nuclei (DNA) and low-speed supernatant egg extract (LSS) and present detailed procedures for sample preparation and application onto grids for negative stain electron microscopy (NS-EM) and cryoelectron microscopy (cryo-EM). For complete details on the use and execution of this protocol, please refer to Cvetkovic et al.1.
RESUMO
The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Šresolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a "closed" conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an "open," nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.