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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 mouse MCM2-7 in vitro with recombinant proteins

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Ivar Ilves

LAB HEAD: Ivar Ilves

PROVIDER: PXD049049 | Pride | 2025-03-31

REPOSITORIES: Pride

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			Action	DRS
	Mm1.raw	Raw
	Mm2.raw	Raw
	allSpectra.HCD.FTMS.secpep.sil0_0.apl	Other
	allSpectra.HCD.FTMS.secpep.sil0_1.apl	Other
	allSpectra.HCD.FTMS.secpep.sil0_10.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 Drosophila CMG in vitro with recombinant proteins

2025-03-31 | PXD049048 | 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

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

Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins.

Project description:MCM2-7 proteins provide essential helicase functions in eukaryotes at chromosomal DNA replication forks. During the G1 phase of the cell cycle, they remain loaded on DNA but are inactive. We have used recombinant methods to show that the Drosophila MCM2-7 helicase is activated in complex with Cdc45 and the four GINS proteins (CMG complex). Biochemical activities of the MCM AAA+ motor are altered and enhanced through such associations: ATP hydrolysis rates are elevated by two orders of magnitude, helicase activity is robust on circular templates, and affinity for DNA substrates is improved. The GINS proteins contribute to DNA substrate affinity and bind specifically to the MCM4 subunit. All pairwise associations among GINS, MCMs, and Cdc45 were detected, but tight association takes place only in the CMG. The onset of DNA replication and unwinding may thus occur through allosteric changes in MCM2-7 affected by the association of these ancillary factors.

| S-EPMC6396293 | biostudies-literature

Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome.

Project description:Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN-MCM-GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD's DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N-Gins51C-GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

| S-EPMC9023282 | biostudies-literature

Origin single-stranded DNA releases Sld3 protein from the Mcm2-7 complex, allowing the GINS tetramer to bind the Mcm2-7 complex.

Project description:The replication fork helicase in eukaryotic cells is comprised of Cdc45, Mcm2-7, and GINS (CMG complex). In budding yeast, Sld3, Sld2, and Dpb11 are required for the initiation of DNA replication, but Sld3 and Dpb11 do not travel with the replication fork. Sld3 and Cdc45 bind to early replication origins during the G(1) phase of the cell cycle, whereas Sld2, GINS, polymerase ε, and Dpb11 form a transient preloading complex that associates with origins during S phase. We show here that Sld3 binds tightly to origin single-stranded DNA (ssDNA). CDK-phosphorylated Sld3 binds to origin ssDNA with similar high affinity. Origin ssDNA does not disrupt the interaction between Sld3 and Dpb11, and origin ssDNA does not disrupt the interaction between Sld3 and Cdc45. However, origin ssDNA substantially disrupts the interaction between Sld3 and Mcm2-7. GINS and Sld3 compete with one another for binding to Mcm2-7. However, in a mixture of Sld3, GINS, and Mcm2-7, origin ssDNA inhibits the interaction between Sld3 and Mcm2-7, whereas origin ssDNA promotes the association between GINS and Mcm2-7. We also show that origin single-stranded DNA promotes the formation of the CMG complex. We conclude that origin single-stranded DNA releases Sld3 from Mcm2-7, allowing GINS to bind Mcm2-7.

| S-EPMC3099676 | biostudies-literature

DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7.

Project description:The committed step of eukaryotic DNA replication occurs when the pairs of Mcm2-7 replicative helicases that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45, GINS, and likely additional proteins are recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). DDK phosphorylation of multiple phosphorylation sites on the Mcm2-7 tails modulates the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity increases the frequency of CMG formation. Our findings reveal the molecular mechanisms sensitizing helicase activation to DDK levels with implications for control of replication origin efficiency and timing.

| S-EPMC7954526 | biostudies-literature

Proximity-based activation of AURORA A by MPS1 potentiates error correction

Project description:Faithfull cell division relies on mitotic chromosomes becoming bioriented with each pair of sister kinetochores bound to microtubules from opposing spindle poles. Erroneous kinetochore- microtubule attachments often form during early mitosis, but are destabilized through the phosphorylation of outer kinetochore proteins by centromeric AURORA B kinase (ABK) and centrosomal AURORA A kinase (AAK), thus allowing for re-establishment of attachments until biorientation is achieved. MPS1-mediated phosphorylation of NDC80 has also been shown to directly weaken the kinetochore-microtubule interface in yeast. In human cells, MPS1 has been proposed to transiently accumulate at end-on attached kinetochores and phosphorylate SKA3 to promote microtubule release. Whether MPS1 directly targets NDC80 and/or promotes the activity of AURORA kinases in metazoans remains unclear. Here, we report a novel mechanism involving communication between kinetochores and centrosomes, wherein MPS1 acts upstream of AAK to promote error correction. MPS1 on pole-proximal kinetochores phosphorylates the C-lobe of AAK thereby increasing its activation at centrosomes. This proximity-based activation ensures the establishment of a robust AAK activity gradient that locally destabilizes mal-oriented kinetochores near spindle poles. Accordingly, MPS1 depletion from Drosophila cells causes severe chromosome misalignment and erroneous kinetochore-microtubule attachments, which can be rescued by tethering either MPS1 or constitutively active AAK mutants to centrosomes. Proximity-based activation of AAK by MPS1 also occurs in human cells to promote AAK-mediated phosphorylation of the NDC80 N-terminal tail. These findings uncover an MPS1-AAK cross-talk that is required for efficient error correction, showcasing the ability of kinetochores to modulate centrosome outputs to ensure proper chromosome segregation.

2025-04-03 | PXD056173 | Pride

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