Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes.
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ABSTRACT:
SUBMITTER: Le TT
PROVIDER: S-EPMC8331092 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase, which unwinds the parental DNA duplex at eukaryotic replication forks, is the key regulated step during replication termination but is poorly understood. In budding yeast, the F-box protein Dia2 drives ubiquitylation of the CMG helicase at the end of replication, leading to a disassembly pathway that requires the Cdc48 segregase. The substrate-binding domain of Dia2 comprises leucine-rich repeats, but Dia2 also has a TPR domain at its amino terminus that interacts with the Ctf4 and Mrc1 subunits of the replisome progression complex, which assembles around the CMG helicase at replication forks. Previous studies suggested two disparate roles for the TPR domain of Dia2, either mediating replisome-specific degradation of Mrc1 and Ctf4 or else tethering SCF(Dia2) (SCF [Skp1/cullin/F-box protein]) to the replisome to increase its local concentration at replication forks. Here, we show that SCF(Dia2) does not mediate replisome-specific degradation of Mrc1 and Ctf4, either during normal S phase or in response to replication stress. Instead, the tethering of SCF(Dia2) to the replisome progression complex increases the efficiency of ubiquitylation of the Mcm7 subunit of CMG, both in vitro and in vivo. Correspondingly, loss of tethering reduces the efficiency of CMG disassembly in vivo and is synthetic lethal in combination with a disassembly-defective allele of CDC48. Residual ubiquitylation of Mcm7 in dia2-ΔTPR cells is still CMG specific, highlighting the complex regulation of the final stages of chromosome replication, about which much still remains to be learned.
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Project description:Cell division cycle protein 45 (Cdc45) is required for DNA synthesis during genome duplication, as a component of the Cdc45-MCM-GINS (CMG) helicase. Despite its essential biological function, its biochemical role in DNA replication has remained elusive. Here we report the 2.1-Å crystal structure of human Cdc45, which confirms its evolutionary link with the bacterial RecJ nuclease and reveals several unexpected features that underpin its function in eukaryotic DNA replication. These include a long-range interaction between N- and C-terminal DHH domains, blocking access to the DNA-binding groove of its RecJ-like fold, and a helical insertion in its N-terminal DHH domain, which appears poised for replisome interactions. In combination with available electron microscopy data, we validate by mutational analysis the mechanism of Cdc45 association with the MCM ring and GINS co-activator, critical for CMG assembly. These findings provide an indispensable molecular basis to rationalize the essential role of Cdc45 in genomic duplication.
| S-EPMC4873980 | biostudies-literature
Project description:Protein modification with poly-ubiquitin chains is a crucial process involved in a myriad of cellular pathways. Chain synthesis requires two steps: substrate modification with ubiquitin (priming) followed by repetitive ubiquitin-to-ubiquitin attachment (elongation). RING-type E3 ligases catalyze both reactions in collaboration with specific priming and elongating E2 enzymes. We provide kinetic insight into poly-ubiquitylation during protein quality control by showing that priming is the rate-determining step in protein degradation as directed by the yeast ERAD RING E3 ligases, Hrd1 and Doa10. Doa10 cooperates with the dedicated priming E2, Ubc6, while both E3s use Ubc7 for elongation. Here, we provide direct evidence that Hrd1 uses Ubc7 also for priming. We found that Ubc6 has an unusually high basal activity that does not require strong stimulation from an E3. Doa10 exploits this property to pair with Ubc6 over Ubc7 during priming. Our work not only illuminates the mechanisms of specific E2/E3 interplay in ERAD, but also offers a basis to understand how RING E3s may have properties that are tailored to pair with their preferred E2s.
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Project description:The initiation of eukaryotic DNA replication occurs in two discrete stages: first, the minichromosome maintenance (MCM) complex assembles as a head-to-head double hexamer that encircles duplex replication origin DNA during G1 phase; then, 'firing factors' convert each double hexamer into two active Cdc45-MCM-GINS helicases (CMG) during S phase. This second stage requires separation of the two origin DNA strands and remodelling of the double hexamer so that each MCM hexamer encircles a single DNA strand. Here we show that the MCM complex, which hydrolyses ATP during double-hexamer formation, remains stably bound to ADP in the double hexamer. Firing factors trigger ADP release, and subsequent ATP binding promotes stable CMG assembly. CMG assembly is accompanied by initial DNA untwisting and separation of the double hexamer into two discrete but inactive CMG helicases. Mcm10, together with ATP hydrolysis, then triggers further DNA untwisting and helicase activation. After activation, the two CMG helicases translocate in an 'N terminus-first' direction, and in doing so pass each other within the origin; this requires that each helicase is bound entirely to single-stranded DNA. Our experiments elucidate the mechanism of eukaryotic replicative helicase activation, which we propose provides a fail-safe mechanism for bidirectional replisome establishment.
| S-EPMC6847044 | biostudies-literature
Project description:Chromosome transmission fidelity 4 (Ctf4) is a conserved protein required for DNA replication. In this report, interactions between human Ctf4 (hCtf4) and the replicative helicase containing the cell division cycle 45 (Cdc45)/minichromosome maintenance 2-7 (Mcm2-7)/Go, Ichi, Nii, and San (GINS) (CMG) proteins [human CMG (hCMG) complex] were examined. The hCtf4-CMG complex was isolated following in vitro interaction of purified proteins (hCtf4 plus the hCMG complex), coinfection of Spodoptera frugiperda (Sf9) insect cells with viruses expressing the hCMG complex and hCtf4, and from HeLa cell chromatin after benzonase and immunoprecipitation steps. The stability of the hCtf4-CMG complex depends upon interactions between hCtf4 and multiple components of the hCMG complex. The hCtf4-CMG complex, like the hCMG complex, contains DNA helicase activity that is more salt-resistant than the helicase activity of the hCMG complex. We demonstrate that the hCtf4-CMG complex contains a homodimeric hCtf4 and a monomeric hCMG complex and suggest that the homodimeric hCtf4 acts as a platform linking polymerase ? to the hCMG complex. The role of the hCMG complex as the core of the replisome is also discussed.
| S-EPMC3856782 | biostudies-literature
Project description: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.
| S-EPMC9578012 | biostudies-literature
Project description:The human CMG helicase (Cdc45-MCM-GINS) is a novel target for anti-cancer therapy due to tumor-specific weaknesses in CMG function induced by oncogenic changes and the need for CMG function during recovery from replicative stresses such as chemotherapy. Here, we developed an orthogonal biochemical screening approach and identified selective CMG inhibitors (CMGi) that inhibit ATPase and helicase activities in an ATP-competitive manner at low micromolar concentrations. Structure-activity information and in silico docking indicate that CMGi occupy ATP binding sites and channels within MCM subunits leading to the ATP clefts, which are likely used for ATP/ADP ingress or egress. CMGi inhibit cell growth and DNA replication using multiple molecular mechanisms. CMGi block helicase assembly steps that require ATP binding/hydrolysis by the MCM complex, specifically MCM ring assembly on DNA and GINS recruitment to DNA-loaded MCM hexamers. During S-phase, inhibition of MCM ATP binding/hydrolysis by CMGi causes a 'reverse allosteric' dissociation of Cdc45/GINS from the CMG that destabilizes the replisome and disrupts interactions with Ctf4, Mcm10, and DNA polymerase-α, -δ, -ε, resulting in DNA damage. These novel CMGi are selectively toxic toward tumor cells and define a new class of CMG helicase-targeted anti-cancer compounds with distinct mechanisms of action.
| S-EPMC10441460 | biostudies-literature
Project description:High-resolution structures have not been reported for replicative helicases at a replication fork at atomic resolution, a prerequisite to understanding the unwinding mechanism. The eukaryotic replicative CMG (Cdc45, Mcm2-7, GINS) helicase contains a Mcm2-7 motor ring, with the N-tier ring in front and the C-tier motor ring behind. The N-tier ring is structurally divided into a zinc finger (ZF) sub-ring followed by the oligosaccharide/oligonucleotide-binding (OB) fold ring. Here we report the cryo-EM structure of CMG on forked DNA at 3.9 Å, revealing that parental DNA enters the ZF sub-ring and strand separation occurs at the bottom of the ZF sub-ring, where the lagging strand is blocked and diverted sideways by OB hairpin-loops of Mcm3, Mcm4, Mcm6, and Mcm7. Thus, instead of employing a specific steric exclusion process, or even a separation pin, unwinding is achieved via a "dam-and-diversion tunnel" mechanism that does not require specific protein-DNA interaction. The C-tier motor ring contains spirally configured PS1 and H2I loops of Mcms 2, 3, 5, 6 that translocate on the spirally-configured leading strand, and thereby pull the preceding DNA segment through the diversion tunnel for strand separation.
| S-EPMC7000775 | biostudies-literature
Project description:Protein modification with poly-ubiquitin chains is a crucial process involved in a myriad of cellular pathways. Chain synthesis requires two steps: substrate modification with ubiquitin (priming) followed by repetitive ubiquitin-to-ubiquitin attachment (elongation). RING-type E3 ligases catalyze both reactions in collaboration with specific priming and elongating E2 enzymes. We provide kinetic insight into poly-ubiquitylation during protein quality control by showing that priming is the rate-determining step in protein degradation as directed by the yeast ERAD RING E3 ligases, Hrd1 and Doa10. Doa10 cooperates with the dedicated priming E2, Ubc6, while both E3s use Ubc7 for elongation. Here, we provide direct evidence that Hrd1 also uses Ubc7 also for priming. We found that Ubc6 has an unusually high basal activity that does not require strong stimulation from an E3. Doa10 exploits these properties to pair with Ubc6 over Ubc7 during priming. Our work not only illuminates the mechanisms of specific E2/E3 interplay in ERAD, but also offers a basis to understand how RING E3s may have properties that are tailored to pair with their preferred E2s.
| S-SCDT-EMBOJ-2020-104863 | biostudies-other
Project description:The activation of eukaryotic origins of replication occurs in temporally separated steps to ensure that chromosomes are copied only once per cell cycle. First, the MCM helicase is loaded onto duplex DNA as an inactive double hexamer. Activation occurs after the recruitment of a set of firing factors that assemble two Cdc45-MCM-GINS (CMG) holo-helicases. CMG formation leads to the underwinding of DNA on the path to the establishment of the replication fork, but whether DNA becomes melted at this stage is unknown1. Here we use cryo-electron microscopy to image ATP-dependent CMG assembly on a chromatinized origin, reconstituted in vitro with purified yeast proteins. We find that CMG formation disrupts the double hexamer interface and thereby exposes duplex DNA in between the two CMGs. The two helicases remain tethered, which gives rise to a splayed dimer, with implications for origin activation and replisome integrity. Inside each MCM ring, the double helix becomes untwisted and base pairing is broken. This comes as the result of ATP-triggered conformational changes in MCM that involve DNA stretching and protein-mediated stabilization of three orphan bases. Mcm2 pore-loop residues that engage DNA in our structure are dispensable for double hexamer loading and CMG formation, but are essential to untwist the DNA and promote replication. Our results explain how ATP binding nucleates origin DNA melting by the CMG and maintains replisome stability at initiation.
| S-EPMC9242855 | biostudies-literature