Abstract
The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS (CMG). The Dbf4-Cdc7 kinase phosphorylates Mcm2 in vitro, but the in vivo role for Dbf4-Cdc7 phosphorylation of Mcm2 is unclear. We find that budding yeast Dbf4-Cdc7 phosphorylates Mcm2 in vivo under normal conditions during S phase. Inhibiting Dbf4-Cdc7 phosphorylation of Mcm2 confers a dominant-negative phenotype with a severe growth defect. Inhibiting Dbf4-Cdc7 phosphorylation of Mcm2 under wild-type expression conditions also results in impaired DNA replication, substantially decreased single-stranded formation at an origin, and markedly disrupted interaction between GINS and Mcm2-7 during S phase. In vitro, Dbf4-Cdc7 kinase (DDK) phosphorylation of Mcm2 substantially weakens the interaction between Mcm2 and Mcm5, and Dbf4-Cdc7 phosphorylation of Mcm2 promotes Mcm2-7 ring opening. The extrusion of ssDNA from the central channel of Mcm2-7 triggers GINS attachment to Mcm2-7. Thus, Dbf4-Cdc7 phosphorylation of Mcm2 may open the Mcm2-7 ring at the Mcm2-Mcm5 interface, allowing for single-stranded DNA extrusion and subsequent GINS assembly with Mcm2-7.
Highlights
The Dbf4-Cdc7 kinase activates DNA replication, and the helicase is composed of Cdc45, Mcm2-7, and GINS
Sld3-Mcm2-7 interaction was detectable in wild-type cells in G1 but not S phase; in mutant cells, Sld3-Mcm2-7 interaction was detectable in G1 and S phase. These data suggest that Dbf4-Cdc7 phosphorylation of Mcm2 is required for Sld3 dissociation from Mcm2-7 in S phase
New Findings in this Study—We found that Dbf4-Cdc7 phosphorylates Ser-164 and Ser-170 of Mcm2 in vivo during S phase under normal growth conditions (Fig. 1)
Summary
The Dbf4-Cdc kinase activates DNA replication, and the helicase is composed of Cdc, Mcm, and GINS. Results: Dbf4-Cdc phosphorylation of Mcm is required in vivo for DNA replication, single-stranded DNA accumulation, and GINS-Mcm interaction. Subsequent studies of ding yeast Dbf4-Cdc phosphorylates Mcm in vivo under budding yeast Mcm proteins by Bochman and Schwacha normal conditions during S phase. In the presence of the replication stress agent methyl methanesulfonate (MMS), a growth defect is observed [26, 27] These data led the authors to conclude that Dbf4-Cdc is not required for cell growth under normal conditions, but that Dbf4-Cdc phosphorylation of Mcm may be important during replication stress [26, 27]. We propose that Dbf4-Cdc phosphorylation of Mcm opens the Mcm2-Mcm gate, allowing for the extrusion of singlestranded DNA from the central channel of Mcm; once single-stranded DNA is extruded, Sld releases from Mcm, allowing GINS to bind to Mcm
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