1. Cdc7-Dbf4 Phosphorylates MCM Proteins via a Docking Site-Mediated Mechanism to Promote S Phase Progression 
Yi-Jun Sheu, Bruce Stillman 
Molecular Cell 
Volume 24, Issue 1, Pages (October 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
A Stable Cdc45-MCM Complex Containing Hyperphosphorylated Mcm4
(A) Biochemical fractionation scheme of yeast cells into soluble and chromatin-bound proteins. Strain OAy535 is used here (Aparicio et al., 1997).
(B) A Cdc45-MCM complex exists on S phase chromatin. Left, silver-stained gel of the Cdc45 immunoprecipitate (+) or the 12CA5 immunoprecipitate (−) of SN2 prepared from cells arrested in G1 (α) and S (HU) phases. Right, results of mass spectrometry for the indicated bands.
(C) Cdc45-MCM interaction occurs only on S phase chromatin. Left, immunoblot analysis of SN1 and SN2 from cells arrested in G1 or S phase. Middle, immunoblot of Cdc45 immunoprecipitates. Right, immunoblot of Mcm4 from Cdc45 immunoprecipitates, SN1 and SN2 fractions.
(D) Mcm4 in the Cdc45-MCM complex is hyperphosphorylated. Immunoblot analysis of the Mcm4 mobility shift in the Cdc45 immunoprecipitate after phosphatase treatment.
(E) Mcm4 hyperphosphorylation is cell-cycle regulated. Immunoblot analysis of SN2 samples from the time course of α factor arrest and release. The percent of unbudded cells is shown.
(F) Cdc45-MCM complex formed in S phase contains hyperphosphorylated Mcm4. Immunoblot analysis of Cdc45 immunoprecipitates of SN2 from the time-course samples. The small panel at right shows Mcm4 from a Cdc45 immunoprecipitate and SN2 side by side for comparison.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
CDC7 Is Required for Normal S Phase Progression, Hyperphosphorylation of Mcm4 In Vivo, and Stable Cdc45-MCM Complex Formation
(A) Growth curve of mcm5-bob1 and mcm5-bob1 cdc7Δ cells. Strains YS814, YS819, YS824, and YS828 are used here.
(B) S phase progression of mcm5-bob1 and mcm5-bob1 cdc7Δ cells. Cells were arrested in G1 using α factor, released into fresh YPD medium at room temperature, collected at indicated times, fixed, and analyzed by flow cytometry for DNA content.
(C) Hyperphosphorylation of Mcm4 is not detectable in cdc7Δ cells arrested in HU. Immunoblot analysis of the Mcm4 mobility shift. Each lane contains 8 μg of SN2 proteins.
(D) Cdc45-MCM complex is not stable in cdc7Δ cells. Left panel, SN2 from HU-arrested cells. Each lane contains 8 μg of SN2 proteins. Right panel, Cdc45 immunoprecipitates.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
In Vitro Phosphorylation of Mcm4 Fragments, Using Purified DDK
(A) Above, domain structure of Mcm4. Below, protein substrates for the kinase assay. Amino acid residues of each substrate are indicated. Substrate 7 has seven S/T to A mutations. Substrate 8 has 3×HA tag replacing the NSD. Substrate 9 is derived from substrate 8 by mutating some of the HA sequence to phosphoacceptors. See Figure 6A for N-terminal sequences of substrates 6–9.
(B) Kinase assay of substrates 1–9 in (A). Left, Coomassie blue-stained gel of the kinase assay. Right, autoradiography of the same gel. Each lane presents 10 μl kinase reaction using 15 nM kinase and 0.5 μM substrate. The first lane (−) has no substrate.
(C) Kinase assay of substrates 10–14 in (A). Same condition as in (B).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Processive Phosphorylation of mcm41–333 through a Kinase-Docking Mechanism
(A) Phosphorylation of mcm41–333 as a function of mcm41–333 concentrations. Ten microliters of kinase reaction using indicated concentrations of kinase and substrate is in each lane. ∗∗∗mcm41–333 and ∗mcm41–333, hyperphosphorylated and hypophosphorylated products, respectively. Autophosphorylated Cdc7 and Dbf4 are indicated.
(B) Quantification of hyperphosphorylated and hypophosphorylated forms of mcm41–333 in (A) using Phospho Imager. The Y-axis unit is arbitrary.
(C) The ratio of hyperphosphorylated over hypophosphorylated mcm41–333 as a function of mcm41–333 concentration calculated from (B).
(D) GST pull-down of purified DDK by GST-mcm4 fusions. Immunoblot analysis of HAHis6-Cdc7 using 12CA5 antibody. Five percent of the input DDK (INP) and 30% of pull-down samples using indicated GST fusions as bait were analyzed.
(E) Mcm4175–333 added in trans can interfere with phosphorylation of mcm41–333. Each lane presents 10 μl of kinase reaction using the indicated amount of DDK, mcm41–333 and either mcm41–175 or mcm4175–333 as competitors. Labels are the same as in (A).
(F) Preincubation of DDK and mcm41–333 eliminates the inhibitory effect of mcm4175–333. No preincubation, same conditions as in (E). Preincubation, DDK and mcm41–333 were preincubated at 30°C for 10 min and then on ice for 1 hr before adding the mcm4175–333 fragment.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
NSD Is Important for Normal Cell Growth and S Phase Progression
(A) Complementation of mcm4Δ by empty vector or plasmids carrying indicated MCM4 alleles using plasmid shuffle assay.
(B) Growth curve of cells carrying MCM4, mcm4Δ2–174, mcm4Δ2–145, or mcm21–200-mcm4Δ2–174 as the sole copy of MCM4 on pRS415.
(C) The cold-sensitive phenotype of mcm4Δ2–174 cells. Cells with indicated MCM alleles were streaked on YPD plates and incubated at 25°C for 2 days and 16°C for 10 days.
(D) mcm4Δ2–174 cells accumulate in S phase. Flow cytometry analysis of DNA content in asynchronous cells in log phase and 4 hr after HU arrest.
(E) Cell-cycle progression of MCM4, mcm4Δ2–174, and mcm21–200-mcm4Δ2–175 cells. Cells were arrested in G1 by α factor, released into fresh YPD medium at room temperature, collected at indicated times, and analyzed for DNA content by flow cytometry.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
DDK Phosphorylation Sites at the N Terminus of Mcm4 Are Important In Vivo
(A) Sequences of mcm4 mutants with altered N terminus.
(B) Complementation of mcm4Δ by plasmids carrying MCM4 alleles described in (A).
(C) Cells with indicated MCM4 alleles were streaked on YPD plates and incubated at 25°C and 15°C for 7 days.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Phosphorylation of MCM by DDK May Be Important for Proper Engagement of Cdc45 to the MCM Complex
(A) Overproduction of Cdc45 is detrimental in NSD mutants of MCM4. Cells with indicated MCM4 alleles carrying control vector (pVF1) or GAL1,10::CDC45 (pGAL::CDC45) were streaked on an SC-Ura plate (left) or an SC-Ura+Gal plate (right) and incubated at 30°C for 4 days.
(B) A model for the recruitment of DDK to licensed origins to regulate proper assembly of pre-IC components during S phase. The ordered recruitment of Cdc45, Sld3, and GINS is largely based on a recent publication (Kanemaki and Labib, 2006). Other factors are omitted for simplicity.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions