The Dbf4-Cdc7 kinase promotes Mcm2-7 ring opening to allow for single-stranded DNA extrusion and helicase assembly

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.

Keywords: DNA Helicase; DNA Replication; DNA-binding Protein; Initiation; Kinase; Phosphorylation; Protein-Protein Interaction.

FIGURE 1.

Dbf4-Cdc7 phosphorylates Mcm2 in budding yeast cells under normal growth conditions. A, left panel, antibody raised against Mcm2 (1-160) was used to probe purified Mcm2 protein that was either unphosphorylated, or phosphorylated by Dbf4-Cdc7 (DDK). Western analysis is shown with position of molecular mass markers to the right of the gel. Duplicate experiments are shown. Right panel, antibody raised against phospho-Mcm2 (Mcm2-161-173,164-phosphoserine,170-phosphoserine) was used to probe purified Mcm2 protein that was either unphosphorylated, or phosphorylated by Dbf4-Cdc7 (DDK). Duplicate experiments are shown. B, left panel, antibody raised against Mcm2 (1-160) was used to probe whole cell extracts from unsynchronized wild-type budding yeast cells or budding yeast cells deleted for cdc7 (Δcdc7, mcm5-bob1). Duplicate experiments are shown. Right panel, antibody raised against phospho-Mcm2 (Mcm2-161-173,164-phosphoserine,170-phosphoserine) was used to probe whole cell extracts from wild-type budding yeast cells or budding yeast cells deleted for cdc7 (Δcdc7, mcm5-bob1). Duplicate experiments are shown. C, left panel, antibody raised against Mcm2 (1-160) was used to probe whole cell extracts from budding yeast cells synchronized with α-factor and then released into medium lacking α-factor for the times indicated. The experiment was performed in the absence and presence of 0.1% MMS. Right panel, antibody raised against phospho-Mcm2 (Mcm2-161-173,164-phosphoserine,170-phosphoserine) was used to probe whole cell extracts from budding yeast cells synchronized with α-factor and then released into medium lacking α-factor for the times indicated. The experiment was performed in the absence and presence of 0.1% MMS.

FIGURE 2.

Expression of mcm2-2A results is a severe defect in growth and DNA replication. A, whole cell extracts from cells harboring a plasmid for GALS induction of MCM2-WT, vector, or mcm2-2A (mcm2-S164A, S170A) were examined by Western analysis for Mcm2 (antibody against Mcm2, 1-160), in the absence or presence of 0.15% galactose at 25 °C. B, top panel, cells from A were plated by 10-fold serial dilutions onto agar plates. Bottom panel, similar to top panel, except the cells harbored the mcm5-bob1 genetic mutation. C, whole cell extracts from cells harboring a plasmid for GALS induction of MCM2-WT, vector, or mcm2-2A (mcm2-S164A, S170A) in an mcm2-td (temperature degron) strain were examined by Western analysis for Mcm2 (antibody against Mcm2, 1-160), in the absence of galactose at 25 °C or in the presence of 0.15% galactose at 37 °C. D, top panel, cells from C were plated by 10-fold serial dilutions onto agar plates. Bottom panel, similar to top panel, except the cells harbored the mcm5-bob1 genetic mutation. E, cells from panels C and D at 37 °C in the presence of 0.15% galactose were arrested with α-factor and released into medium lacking α-factor for the time point indicated. Cells were analyzed by FACS with propidium iodine staining for DNA content.

FIGURE 3.

Dbf4-Cdc7 phosphorylation of Mcm2 is required for ssDNA formation at an origin of replication during S phase. Cells from Fig. 2 (C and D) at 37 °C in the presence of 0.15% galactose were arrested with α-factor (G₁ phase cells) and released into medium lacking α-factor for 30 min (S phase cells). Cells were fixed, immunoprecipitated with antibodies directed against RPA, and analyzed by quantitative PCR for DNA region encompassing the early origins ARS305 or ARS306, or the region of DNA midway between ARS305 and ARS306. Error bars indicate S.E.

FIGURE 4.

Dbf4-Cdc7 phosphorylation of Mcm2 is required for GINS-Mcm2-7 interaction during S phase. A, cells from Fig. 2 (C and D) incubated at 37 °C in the presence of 0.15% galactose were arrested with α-factor and released into medium lacking α-factor for the time points indicated. Left panel, whole cell extracts were probed for antibodies directed against Mcm2, Cdc45, Psf2 (a component of GINS), or Sld3. Right panel, co-IP. Unfixed cells were immunoprecipitated with antibodies directed against Mcm2 followed by Western analysis with antibodies directed against Mcm2, Cdc45, Psf2, or Sld3. B, Similar to A, except the indicated cell strains harbored the mcm5-bob1 mutation.

FIGURE 5.

Dbf4-Cdc7 phosphorylation of Mcm2 weakens the interaction between Mcm2 and Mcm5. Purified proteins were used in these experiments. A, purified GST-Cdc45 was used to pull down Dbf4-Cdc7 (DDK)-phosphorylated Mcm2 or PKA-phosphorylated Mcm2. The kinases were removed from the reaction prior to the GST pulldown experiment. Radiolabeled protein was matched for total radioactive counts and total protein prior to analysis. The purified Mcm2 protein harbored an N-terminal PKA tag for radiolabeling with PKA. The PKA tag is not physiologic; it is used as a control. The product of the pulldown was analyzed by SDS-PAGE followed by phosphorimaging quantitation, and plotting. B, similar to A, except GST-Mcm6 was used, and the experiment was performed in the absence or presence of 0.1 mm ATP-γS. C, similar to B, except GST-Mcm5 was used. D, similar to C except GST-Mcm5 wild-type or GST-Mcm5-bob1 was used to pull down PKA-radiolabeled Mcm2. E, similar to D, except GST-Mcm5 wild type or GST-Mcm5-bob1 was used to pull down PKA-radiolabeled Mcm3. Error bars indicate S.E.

FIGURE 6.

Dbf4-Cdc7 phosphorylation of Mcm2 promotes Mcm2-7 ring opening. A, 20 nm wild-type Mcm2-7, Mcm2-7-Mcm2-2D (Mcm2-S164D, S170D), Mcm pentamer (Mcm2-7 lacking Mcm6), or Mcm2-7-Mcm5-bob1 was incubated with 20 nm radiolabeled 1.5-kb linear ssDNA containing the ARS305 origin for varying amount of time. The fraction of DNA bound was calculated with filter binding, plotted, and fit to a logarithmic equation. B, same as A, except circular ssDNA of identical sequence and length was used. C, varying concentrations (2–60 nm) of wild-type Mcm2-7, Mcm2-7-Mcm2-2D (Mcm2-S164D, S170D), Mcm pentamer (Mcm2-7 lacking Mcm6), or Mcm2-7-Mcm5-bob1 were incubated with 2 nm radiolabeled 1.5-kb linear ssDNA containing the ARS305 origin for 10 min. The fraction DNA bound was calculated with filter binding, plotted, and fit to a logarithmic equation. D, same as C, except circular ssDNA of identical sequence and length was used. Error bars indicate S.E.

FIGURE 7.

Model for the function of Dbf4-Cdc7 in the initiation of DNA replication. A, in G₁, Cdc45-Sld3 is bound to Mcm2-7. Sld7 is also bound to Sld3 (not shown). Mcm2-7 exists as a double hexamer in G₁. B, in S phase, Dbf4-Cdc7 phosphorylates Mcm2, Mcm4, and Mcm6. Dbf4-Cdc7 phosphorylation of Mcm4 may be important for Cdc45-Mcm2-7 interaction during S phase because Dbf4-Cdc7 phosphorylation of Mcm4 alleviates an inhibitory activity of an N-terminal region of Mcm4. Dbf4-Cdc7 phosphorylation of Mcm4 and Mcm6 may also be important for double hexamer dissociation. Dbf4-Cdc7 also phosphorylates Mcm2, an activity that opens the Mcm2-Mcm5 gate, allowing single-stranded DNA to be extruded from the central channel of Mcm2-7. Sld3 dissociates from Mcm2-7 and grips onto the extruded single strand of DNA. Sld2 is bound to Sld3 during S phase, and Sld2 may similarly transition from binding Mcm2-7 in G₁ to binding single-stranded DNA during S phase. C, GINS binds to the Mcm2-7 and Cdc45, forming the CMG closed-ring complex encircling single-stranded DNA.