In preparation for bidirectional DNA replication, the origin recognition complex (ORC) loads two hexameric MCM helicases to form a head-to-head double hexamer around DNA
. The mechanism of MCM ...double-hexamer formation is debated. Single-molecule experiments have suggested a sequential mechanism, in which the ORC-dependent loading of the first hexamer drives the recruitment of the second hexamer
. By contrast, biochemical data have shown that two rings are loaded independently via the same ORC-mediated mechanism, at two inverted DNA sites
. Here we visualize MCM loading using time-resolved electron microscopy, and identify intermediates in the formation of the double hexamer. We confirm that both hexamers are recruited via the same interaction that occurs between ORC and the C-terminal domains of the MCM helicases. Moreover, we identify the mechanism of coupled MCM loading. The loading of the first MCM hexamer around DNA creates a distinct interaction site, which promotes the engagement of ORC at the N-terminal homodimerization interface of MCM. In this configuration, ORC is poised to direct the recruitment of the second hexamer in an inverted orientation, which is suitable for the formation of the double hexamer. Our results therefore reconcile the two apparently contrasting models derived from single-molecule experiments and biochemical data.
Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, ...Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro.
Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM ...phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.
Eukaryotic origins of replication are licensed upon loading of the MCM helicase motor onto DNA. ATP hydrolysis by MCM is required for loading and the post-catalytic MCM is an inactive double hexamer ...that encircles duplex DNA. Origin firing depends on MCM engagement of Cdc45 and GINS to form the CMG holo-helicase. CMG assembly requires several steps including MCM phosphorylation by DDK. To understand origin activation, here we have determined the cryo-EM structures of DNA-bound MCM, either unmodified or phosphorylated, and visualize a phospho-dependent MCM element likely important for Cdc45 recruitment. MCM pore loops touch both the Watson and Crick strands, constraining duplex DNA in a bent configuration. By comparing our new MCM-DNA structure with the structure of CMG-DNA, we suggest how the conformational transition from the loaded, post-catalytic MCM to CMG might promote DNA untwisting and melting at the onset of replication.
Kinesin-5 motors are vital mitotic spindle components, and disruption of their function perturbs cell division. We investigated the molecular mechanism of the human kinesin-5 inhibitor GSK-1, which ...allosterically promotes tight microtubule binding. GSK-1 inhibits monomeric human kinesin-5 ATPase and microtubule gliding activities, and promotes the motor's microtubule stabilization activity. Using cryoelectron microscopy, we determined the 3D structure of the microtubule-bound motor-GSK-1 at 3.8 Å overall resolution. The structure reveals that GSK-1 stabilizes the microtubule binding surface of the motor in an ATP-like conformation, while destabilizing regions of the motor around the empty nucleotide binding pocket. Density corresponding to GSK-1 is located between helix-α4 and helix-α6 in the motor domain at its interface with the microtubule. Using a combination of difference mapping and protein-ligand docking, we characterized the kinesin-5-GSK-1 interaction and further validated this binding site using mutagenesis. This work opens up new avenues of investigation of kinesin inhibition and spindle perturbation.
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•GSK-1 inhibits human kinesin-5 monomers thereby promoting microtubule stabilization•GSK-1 binds between helix-α4 and helix-α6 at the motor-microtubule interface•GSK-1 traps the kinesin-5 microtubule binding interface in an ATP-like state•GSK-1 perturbs the nucleotide binding site thereby preventing ATP binding
To investigate mitotic kinesin inhibition mechanisms, we used cryo-EM to determine the 3.8-Å resolution structure of human kinesin-5 trapped on the microtubule by the drug GSK-1. GSK-1 binds between helix-α4 and helix-α6 in the motor domain, stabilizing the motor's microtubule binding interface while allosterically preventing nucleotide binding.
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.
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•Vertical DNA movement through the MCM ring requires rotation inside the pore•Structural asymmetries in MCM-DNA are captured during ATPase-powered translocation•Asymmetric rotation explains selective ATPase site requirements for translocation•The fork-stabilization complex strengthens parental-DNA engagement by the MCM
Eickhoff et al. used cryo-EM to image DNA unwinding by the eukaryotic replicative helicase, Cdc45-MCM-GINS. As the hexameric MCM ring hydrolyses ATP, DNA is spooled asymmetrically around the ring pore. This asymmetry explains why selected ATPase sites are essential for DNA translocation. Understanding DNA unwinding informs on replication fork progression.
Kinesin motors play diverse roles in mitosis and are targets for antimitotic drugs. The clinical significance of these motors emphasizes the importance of understanding the molecular basis of their ...function. Equally important, investigations into the modes of inhibition of these motors provide crucial information about their molecular mechanisms. Kif18A regulates spindle microtubules through its dual functionality, with microtubule-based stepping and regulation of microtubule dynamics. We investigated the mechanism of Kif18A and its inhibition by the small molecule BTB-1. The Kif18A motor domain drives ATP-dependent plus-end microtubule gliding, and undergoes conformational changes consistent with canonical mechanisms of plus-end–directed motility. The Kif18A motor domain also depolymerizes microtubule plus and minus ends. BTB-1 inhibits both of these microtubule-based Kif18A activities. A reconstruction of BTB-1–bound, microtubule-bound Kif18A, in combination with computational modeling, identified an allosteric BTB-1–binding site near loop5, where it blocks the ATP-dependent conformational changes that we characterized. Strikingly, BTB-1 binding is close to that of well-characterized Kif11 inhibitors that block tight microtubule binding, whereas BTB-1 traps Kif18A on the microtubule. Our work highlights a general mechanism of kinesin inhibition in which small-molecule binding near loop5 prevents a range of conformational changes, blocking motor function.
Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of ...intracellular cargo distribution. Kinesin-binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment, and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a tetratricopeptide repeat-containing, right-handed α-solenoid that sequesters the kinesin motor domain's tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.
Fluorescence spectroscopy provides an excellent technique for investigating heterogeneous systems, due to its high sensitivity and the large effect of the local environment on molecular emission. In ...addition, the use of polarity-sensitive fluorescent probes as guests in supramolecular host–guest inclusion complexes can be exploited in fluorescent sensors. This paper identifies, tabulates, and quantifies a series of useful polarity-sensitive fluorescent probes, with a wide range of polarity-dependent fluorescence responses. The degree of polarity sensitivity is quantified using the polarity sensitivity factor (PSF), developed in our laboratory. In most cases, such polarity-sensitive probes show increased emission as the local polarity is decreased (PSF > 1); 10 such probes are described. However, less commonly, “reverse polarity dependence” can occur in which probe emission decreases with decreasing polarity (PSF < 1); four such probes are described. The mechanism for the observed polarity-induced fluorescence changes will also be discussed in selected representative cases. The purpose of this paper is to present details on a broad arsenal of polarity-sensitive fluorescence probes with varying properties, with potentially useful applications in the study of heterogeneous systems, including inclusion phenomena, and in practical applications such as fluorescent sensors, which will be useful to researchers studying supramolecular and other heterogeneous systems using fluorescence spectroscopy.
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 Å resolution 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.
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•The γ-tubulin ring complex (γTuRC) nucleates microtubules and caps their minus ends•Microtubule nucleation from purified γTuRC is highly cooperative, yet inefficient•A partly open, asymmetric structure of γTuRC explains inefficient nucleation•Actin and MZT2 stabilize the closed part of the γTuRC structure
Consolati et al. find that microtubule nucleation by individual γTuRC complexes is inefficient despite its proposed role as a nucleating template. A 4 Å structure of the complex reveals a mismatch with the microtubule structure, explaining the inefficiency of nucleation and providing a possible mechanism for the regulation of nucleation.
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