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Results for “CMG Helicase”

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5 recordsLinked to original sources
PubMed2026

MCM10 and RECQL4 have cooperative and redundant roles in activating the CMG helicase during the replication initiation.

DNA replication initiation requires activation of the CMG helicase to establish the replisome. This process involves the extrusion of single-stranded DNA (ssDNA) from the central channel of MCM double hexamers, allowing the two CMG helicases to pass each other; however, the factors that mediate this process in human cells remain unclear. We show that degron-mediated depletion of either MCM10 or RECQL4 alone causes mild replication defects, whereas simultaneous depletion of both proteins severely impairs CMG activation. ChIP-seq analyses demonstrate that RECQL4 localizes to replication initiation zones (IZs) independently of MCM10, whereas MCM10 recruitment to IZs is enhanced upon RECQL4 depletion, consistent with partially redundant roles during CMG activation. Rescue experiments further indicate that RECQL4 cooperates with MCM10 through direct interaction, and that their ssDNA-binding activity underlies their functional overlap. We propose that MCM10 and RECQL4 act cooperatively and redundantly to promote CMG activation.

CMG activation↗
PubMed2026

Direct visualization of MCM helicase activation and replisome coupling in situ.

Deciphering the spatial organization of molecular machines that copy the genome remains a fundamental challenge in biology. Essential for eukaryotic DNA replication, Mini-Chromosome Maintenance (MCM2-7) helicases are loaded during G1 as double hexamers (DHs) to license replication origins. Upon activation in S phase, each DH is thought to split into two single hexamers (SHs) that form the active CMG helicases and travel bidirectionally. However, the field has long been divided: biochemical and structural studies define CMG helicases as autonomous, independent motors, while genomic and cellular imaging assays suggest sister replisomes remain physically coupled within replication factories. Here, we use MINFLUX nanoscopy to localize individual MCM complexes down to nanometer precision in situ, directly resolving DHs in human cells and capturing their separation into SHs upon origin firing. We find that the resulting sister replisomes do not diffuse apart: they remain coupled at a characteristic distance of ~40 nm throughout S phase. Depletion experiments identify two distinct contributions to this coupling: local, protein-mediated tethering by the AND1 scaffold, and higher-order spatial confinement dependent on cohesin, which is dispensable for MCM loading in G1 but required to maintain coupling in S phase. By linking the nanometer-scale architecture of the replisome to the genome-wide topology of replication fountains, these findings provide direct spatial evidence that sister forks are coupled during DNA synthesis and define the molecular forces that organize replisomes within their native nuclear context.

DNA replication↗
PubMed2025

The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks.

Understanding how DNA replication forks stall and restart and how the DNA replication checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here, we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading-strand DNA synthesis quickly stops but CMG helicase continues to unwind, and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC, and DNA polymerases δ and ε, which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA replication checkpoint restrains fork progression, which limits this sequestration, protecting stalled forks from collapse and ensuring restart.

DNA Replication↗
PubMed2025

MCM5 UFMylation regulates replication origin firing and fork progression.

Modification with UFM1 (UFMylation) is essential for cell proliferation, but its precise mechanism of action is unclear. Furthermore, the UFMylation pathway has been associated with microcephalic primordial dwarfism (MPD) disorders, and mutations causative for MPD are also identified in genes encoding components of the replicative DNA helicase complex, including the MCM hexamer. Here, we reveal that UFMylation regulates DNA replication, and that all MPD-associated mutations in UFMylation enzymes impair replication. Mechanistically, the UFM1 E3 ligase UFL1 catalyzes Lys583 UFMylation of MCM5, a critical component of the CMG replicative DNA helicase complex. Mutation of Lys583 blocking this UFMylation event destabilizes the helicase complex, delaying origin firing and slowing replication fork progression. We conclude that MCM5 UFMylation is essential for efficient origin firing and replication fork progression, both of which ensure accurate DNA replication, cell proliferation, and prevention of MPD disorders.

DNA Replication↗
PubMed2026

Structural insights into Sld3-Sld7-dependent Cdc45 loading during replication initiation.

Regulated helicase activation by DDK kinase is central for genome stability. However, how DDK phosphorylation primes the MCM2-7 double hexamer (DH) for Sld3-Sld7 binding and Cdc45 loading remained unclear. We define this mechanism through cryo-EM structures of MCM2-7 DH-Sld3-Sld7 (MS) and MCM2-7 DH-Sld3-Sld7-Cdc45 (MSC). We reveal that the autoinhibitory Mcm4 tail engages not only Mcm4 but also Mcm6. Upon DDK-dependent phosphorylation, both of these sites become accessible. In the context of the MS structure, we identify that two short Sld3 motifs that contact Mcm4 and Mcm6 read out the DH phosphorylation state, while the Sld3 Treslin domain (STD) binds to Mcm2. In the MSC structure, Cdc45 dislodges the Sld3 STD from Mcm2, allowing Sld3 to position Cdc45 at the Mcm2/Mcm5 interface. Mutagenesis of the Sld3 STD-Cdc45 interface disrupts Cdc45 loading, validating this interaction. Together, our data reveal a phosphorylation-encoded mechanism coupling DDK-activated Mcm4/Mcm6 surfaces to distal Cdc45 placement, explaining how firing factors choreograph the DH-to-CMG transition.

Cell Cycle Proteins↗