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Conserved phosphorylation mechanism regulates the interaction between the Cdc45-MCM2-7-GINS (CMG) replicative helicase complex and its forked DNA substrate

ABSTRACT: Mapping of the Chk1 phosphorylation sites in Drosophila CMG in vitro with recombinant proteins

INSTRUMENT(S): LTQ Orbitrap, Q Exactive

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

SUBMITTER: Ivar Ilves

LAB HEAD: Ivar Ilves

PROVIDER: PXD049048 | Pride | 2025-03-31

REPOSITORIES: Pride

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			Action	DRS
	Dm1.raw	Raw
	Dm2.raw	Raw
	Dmel_Chk1_CMG_uniprotKB_2023_09_25.fasta	Fasta
	allSpectra.HCD.FTMS.secpep.sil0_0.apl	Other
	allSpectra.HCD.FTMS.secpep.sil0_1.apl	Other

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Publications

A conserved phosphorylation mechanism for regulating the interaction between the CMG replicative helicase and its forked DNA substrate.

Koit Sandra S Tamberg Nele N Reinapae Allan A Peil Lauri L Kristjuhan Arnold A Ilves Ivar I

The Journal of biological chemistry 20250314 4

The CMG helicase is a crucial enzyme complex that plays a vital role in the replication of genomic DNA in eukaryotes. Besides unwinding the DNA template and coordinating the replisome's structure, it is also a key target for signaling pathways that regulate the replication process. We show that a specific serine/threonine residue in the MCM3 subunit of CMG, which has been previously linked to phosphorylation-dependent control mechanisms of genomic DNA replication in human cells, is a conserved p ...[more]

PMID: 40090586

Similar Datasets

Conserved phosphorylation mechanism regulates the interaction between the Cdc45-MCM2-7-GINS (CMG) replicative helicase complex and its forked DNA substrate

Project description:Mapping of the Chk1 phosphorylation sites in mouse MCM2-7 in vitro with recombinant proteins

2025-03-31 | PXD049049 | Pride

Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase.

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 α to the hCMG complex. The role of the hCMG complex as the core of the replisome is also discussed.

| S-EPMC3856782 | biostudies-literature

Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex.

Project description:The replication of eukaryote chromosomes slows down when DNA is damaged and the proteins that work at the fork (the replisome) are known targets for the signaling pathways that mediate such responses critical for accurate genomic inheritance. However, the molecular mechanisms and details of how this response is mediated are poorly understood. In this report we show that the activity of replisome helicase, the Cdc45/MCM2-7/GINS (CMG) complex, can be inhibited by protein phosphorylation. Recombinant Drosophila melanogaster CMG can be stimulated by treatment with phosphatase whereas Chk2 but not Chk1 interferes with the helicase activity in vitro. The targets for Chk2 phosphorylation have been identified and reside in MCM subunits 3 and 4 and in the GINS protein Psf2. Interference requires a combination of modifications and we suggest that the formation of negative charges might create a surface on the helicase to allosterically affect its function. The treatment of developing fly embryos with ionizing radiation leads to hyperphosphorylation of Psf2 subunit in the active helicase complex. Taken together these data suggest that the direct modification of the CMG helicase by Chk2 is an important nexus for response to DNA damage.

| S-EPMC3421224 | biostudies-literature

Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase.

Project description:The protein Cdc45 plays a critical but poorly understood role in the initiation and elongation stages of eukaryotic DNA replication. To study Cdc45's function in DNA replication, we purified Cdc45 protein from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps and found that the protein exists in a stable, high-molecular-weight complex with the Mcm2-7 hexamer and the GINS tetramer. The purified Cdc45/Mcm2-7/GINS complex is associated with an active ATP-dependent DNA helicase function. RNA interference knock-down experiments targeting the GINS and Cdc45 components establish that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core helicase machinery for eukaryotic DNA replication.

| S-EPMC1482467 | biostudies-literature

DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in C. elegans

Project description:Assembly of the DNA helicase known as CMG (CDC45-MCM-GINS) is the key regulated step during DNA replication initiation in eukaryotes. Using the Caenorhabditis elegans embryo as a model system, we identify a new CMG assembly factor called DNSN-1, which associates with the BRCT-domain protein MUS-101. We show that DNSN-1 is required to recruit the GINS complex to chromatin and find that DNSN-1 positions GINS on the MCM-2-7 helicase motor, by direct binding of DNSN-1 to GINS and MCM-3, on interfaces that are important for initiation and essential for viability.

2023-10-24 | PXD044240 | Pride

The Mcm2-Ctf4-Pola axis facilitates parental histone transfer to lagging strands

Project description:Although essential for epigenetic inheritance, the transfer of parental histone (H3-H4)2 tetramers that contain epigenetic modifications to replicating DNA strands is poorly understood. Here, we show that the Mcm2-Ctf4-Pol axis facilitates the transfer of parental (H3-H4)2 tetramers to lagging strands of DNA replication forks. Mutating the H3-H4 binding domain of Mcm2, a subunit of the CMG (Cdc45-MCM-GINS) replicative helicase that translocates along leading strands, impairs the transfer of parental (H3-H4)2 to lagging strands. Similar effects are observed in Ctf4 and Pol-primase mutants that disrupt the connection of the CMG helicase via Ctf4-Pol to lagging strands. Our results support a model whereby parental (H3-H4)2 displaced from nucleosomes through leading-strand DNA replication are transferred to lagging strands for nucleosome assembly via the CMG-Ctf4-Pol complex.

2018-09-24 | GSE118580 | GEO

CRL2LRR1 is required for CMG unloading during vertebrate replication termination

Project description:A key event during eukaryotic replication termination is the removal of the CMG helicase from chromatin. CMG unloading involves ubiquitylation of its MCM7 subunit and the action of the p97 ATPase. Using a proteomic screen in Xenopus egg extracts, we identified factors that are retained on chromatin when CMG unloading is blocked and identified the E3 ubiquitin ligase CRL2LRR1 and several other potential regulators of termination including a specific p97 complex. We show that CRL2LRR1 recruitment coincides with MCM7 ubiquitylation and occurs only when replisomes converge. In the absence of CRL2LRR1, MCM7 is not ubiquitylated, CMG unloading is inhibited, and many replisome components including DNA pol are retained on DNA. Our data identify CRL2LRR1 as a master regulator of replisome disassembly during vertebrate DNA replication termination.

2017-02-02 | PXD004828 | Pride

Mcm7 ubiquitylation and Pif1-family helicases drive CMG disassembly to preserve genome integrity

Project description:Disassembly of the DNA helicase known as CMG (Cdc45-Mcm-GINS) is the key regulated step during DNA replication termination in eukaryotes. In budding yeast, CMG disassembly is initiated by the ubiquitin ligase SCF-Dia2, which ubiquitylates the Mcm7 subunit of CMG, leading to recruitment of the Cdc48 unfoldase. Here we have investigated the partners of the F-box protein Dia2 and tested which ones are dependent upon the TPR domain of Dia2, using yeast strains expressing ProteinA-tagged Dia2 or Dia2∆TPR. Controls include a strain expressing the TAP tag alone, and a strain expressing TAP-tagged Sld5 subunit of the CMG helicase.

2025-04-09 | PXD048935 | Pride

The structural basis for MCM2-7 helicase activation by GINS and Cdc45.

Project description:Two central steps for initiating eukaryotic DNA replication involve loading of the Mcm2-7 helicase onto double-stranded DNA and its activation by GINS-Cdc45. To better understand these events, we determined the structures of Mcm2-7 and the CMG complex by using single-particle electron microscopy. Mcm2-7 adopts two conformations--a lock-washer-shaped spiral state and a planar, gapped-ring form--in which Mcm2 and Mcm5 flank a breach in the helicase perimeter. GINS and Cdc45 bridge this gap, forming a topologically closed assembly with a large interior channel; nucleotide binding further seals off the discontinuity between Mcm2 and Mcm5, partitioning the channel into two smaller pores. Together, our data help explain how GINS and Cdc45 activate Mcm2-7, indicate that Mcm2-7 loading may be assisted by a natural predisposition of the hexamer to form open rings, and suggest a mechanism by which the CMG complex assists DNA strand separation.

| S-EPMC4184033 | biostudies-literature

Aspergillus affinis strain:CMG 70

Project description:Aspergillus affinis strain:CMG 70 Genome sequencing and assembly

| PRJNA723818 | ENA