Mec1 is one of multiple kinases that prime the Mcm2-7 helicase for phosphorylation by Cdc7

Area of Science:

• Molecular Biology
• Cell Cycle Regulation
• DNA Replication

Background:

• The eukaryotic replicative DNA helicase, Mcm2-7 complex, requires Dbf4-dependent kinase (DDK) for activation.
• DDK-mediated phosphorylation of Mcm2-7 is dependent on prior phosphorylation by unknown kinases.

Purpose of the Study:

• To identify the priming phosphorylation sites on Mcm2-7 required for DDK activation.
• To elucidate the role of these priming phosphorylations in DNA replication and cell cycle progression.

Main Methods:

• Phosphorylation site analysis of Mcm4 and Mcm6 subunits.
• Site-directed mutagenesis, including phosphomimetic mutations.
• Genetic interaction studies with replication fork stability factors (MRC1, TOF1).

Main Results:

• Identified DDK phosphorylation sites on Mcm4 and Mcm6; phosphorylation of either is sufficient for proliferation.
• Demonstrated that priming phosphorylation of S/T-P or S/T-Q motifs is essential for DDK phosphorylation and S phase passage.
• Phosphomimetic mutations bypassed DDK function and priming site mutations.
• Mrc1 facilitates Mec1 phosphorylation of S/T-Q motifs for replication activation.

Conclusions:

• Discovered essential priming phosphorylation events regulating Mcm2-7 helicase activation.
• Established the necessity of these modifications for normal S phase progression and replication fork stability.
• Revealed regulatory mechanisms controlling origin firing and replication fork assembly.