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Structural Insight into the MCM double hexamer activation by Dbf4-Cdc7 kinase
Jiaxuan Cheng1,2, Ningning Li1, Yunjing Huo3
1State Key Laboratory of Membrane Biology, Peking-Tsinghua Joint Center for Life Sciences, School of Life Sciences, Peking University, Beijing, 100871, China.
Abstract:
The Dbf4-dependent kinase Cdc7 (DDK) regulates DNA replication initiation by phosphorylation of the MCM double hexamer (MCM-DH) to promote helicase activation. Here, we determine a series of cryo electron microscopy (cryo-EM) structures of yeast DDK bound to the MCM-DH. These structures, occupied by one or two DDKs, differ primarily in the conformations of the kinase core. The interactions of DDK with the MCM-DH are mediated exclusively by subunit Dbf4 straddling across the hexamer interface on the three N-terminal domains (NTDs) of subunits Mcm2, Mcm6, and Mcm4. This arrangement brings Cdc7 close to its only essential substrate, the N-terminal serine/threonine-rich domain (NSD) of Mcm4. Dbf4 further displaces the NSD from its binding site on Mcm4-NTD, facilitating an immediate targeting of this motif by Cdc7. Moreover, the active center of Cdc7 is occupied by a unique Dbf4 inhibitory loop, which is disengaged when the kinase core assumes wobbling conformations. This study elucidates the versatility of Dbf4 in regulating the ordered multisite phosphorylation of the MCM-DH by Cdc7 kinase during helicase activation.
Insights
The Dbf4-dependent kinase Cdc7 (DDK) regulates DNA replication initiation. Cryo-EM structures reveal how Dbf4 binding to the MCM double hexamer (MCM-DH) positions Cdc7 for essential phosphorylation events, controlling helicase activation.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- DNA replication initiation is a critical cell process regulated by kinases.
- The Dbf4-dependent kinase Cdc7 (DDK) phosphorylates the MCM double hexamer (MCM-DH) to activate DNA helicase.
- Understanding DDK-MCM interactions is key to deciphering replication control.
Purpose of the Study:
- To determine the structural basis of DDK-MCM-DH interaction.
- To elucidate the mechanism of MCM helicase activation by DDK.
- To visualize the role of Dbf4 in substrate targeting and kinase regulation.
Main Methods:
- Cryo-electron microscopy (cryo-EM) to obtain high-resolution structures.
- Biochemical assays to study kinase activity and substrate interaction.
- Structural analysis of DDK bound to MCM-DH.
Main Results:
- Cryo-EM structures show yeast DDK bound to MCM-DH in different conformations.
- Dbf4 subunit mediates DDK interaction with MCM-DH N-terminal domains (NTDs).
- Dbf4 positions Cdc7 kinase to phosphorylate Mcm4's N-terminal serine/threonine-rich domain (NSD) and disengages an inhibitory loop.
Conclusions:
- Dbf4 acts as a versatile regulator, bridging DDK to MCM-DH and facilitating targeted phosphorylation.
- Structural insights reveal how Dbf4 controls Cdc7 kinase activity and substrate access.
- This work clarifies the mechanism of MCM helicase activation during DNA replication initiation.
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