Supplemental Material

This article contains the following supporting material:

• Figure 1 - (A) One μg of bacterially-expressed purified His-MCM2M5 were incubated with baculovirus-expressed purified 50 ng Cdc7/Dbf4 or Cdc7kd/Dbf4 in the presence of γ-³²PATP. Kinase reaction was resolved by SDS/PAGE and visualized by autoradiography (left, upper panel) or Coomassie blue staining (left, lower panel). MCM2M5 bands were then cut out from gel and their radioactivities were measured using a scintillation counter (right). (B). Summary of Cdc7/Dbf4 phosphorylation sites in human MCM2. Shown are schematic representations of human MCM2 structural domains, its N-terminal amino sequence (MCM2M5), major in vivo phosphorylation sites and Cdc7/Dbf4 in vitro phosphorylation sites, Ser27, Ser41 and Ser139 (red) and minor in vivo phosphorylation sites, Ser53 and Ser108 (blue).

• Figure 2 - (A) MS/MS spectrum of the peptide GNDPLTSpSPGR. Predicted masses for type b and type y ions are listed above and below the sequence, respectively. Observed masses are underlined. Note that y₃, y₄, y₅, and phosphate losses from y₄ and y₅ are detected, definitively identifying residue 8 as phosphoserine. *, loss of water/ammonia from y or b ion. (B) MS/MS spectrum of the peptide TDALTSpSPGR. Predicted masses for type b and type y ions are listed above and below the sequence, respectively. Observed masses are underlined. Note that y₃, y₄, y₅, and phosphate losses from y4 and y5 are detected, definitively identifying residue 7 as phosphoserine. *, loss of water/ammonia from y or b ion. (C) MS/MS spectrum of the peptide GLLYDpSDEEDEERPAR. Predicted masses for type b and type y ions are listed above and below the sequence, respectively. Observed singly, doubly, and triply charged product ions are indicated by single, double, and triple underlines, respectively. Note that y₁₀²⁺, y₁₁²⁺, and y₁₁²⁺ - phosphate are detected, definitively identifying residue 6 as phosphoserine. (D) MCM2M5 and its point mutant proteins, in which Ser27, Ser41, Ser139, all three, Ser26 and Ser139, or Ser40 and Ser139 were substituted by Ala (MCM2M5S27A, MCM2M5S41A, MCM2M5S139A, MCM2M5A, MCM2M5S26AS139A or MCM2M5S40AS139A) were phosphorylated by purified Cdc7/Dbf4 in vitro in the presence of γ-³²PATP. Proteins were separated by SDS/PAGE, eluted from the gels, digested with trypsin and subjected to 2-D tryptic phosphopeptide mapping analysis as in Figure 1. Shown are 2-D maps of (a) MCM2M5 (b) MCM2M5S27A, (c) MCM2M5S41A, (d) MCM2M5S139A, (e) MCM2M5A, (f) MCM2M5S26AS139A and (g) MCM2M5S40AS139A. A schematic map is shown in (h). Note: MCM2M5A was very weakly phosphorylated (nonspecifically) by purified Cdc7/Dbf4 in vitro. Therefore, very little ³²P-lebeled MCM2M5A was recovered from in vitro phosphorylation assay and used in 2-D mapping analysis.

• Figure 3 - α-MCM2S1 specifically recognizes Cdc7/Dbf4 phosphorylated MCM2 Ser139. (A) HeLa cell lysates were subjected to SDS/PAGE, transferred into the PVDF membrane and immunoblotted with α-MCM2S1 (left) and α-MCM2M1 (right). Immunoblotting analysis indicated that α-MCM2M1 or α-MCM2S1 detected ~120 kDa endogenous MCM2 protein in HeLa cells. (B) HeLa cell lysates were immunoprecipitated by α-MCM2M1 and the immunoprecipitates were treated with or without λ phosphatase (λpase) or alkaline phosphatase (APase) at 30 0C for 30 min. The reactions stopped by addition of sample buffer were subjected to SDS/PAGE, transferred to the PVDF membrane and immunoblotted with α-MCM2S1 (upper panel) and α-MCM2M1 (lower panel). (C) HeLa cells transfected with indicated plasmids were lyzed and cell lysates were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates were subjected to SDS/PAGE, transferred to the PVDF membrane and immunoblotted with α-MCM2S1 (upper panel) or α-MCM2M1 (lower panel). (D) HeLa cell lysates were subjected into SDS/PAGE, transferred to PVDF membrane and immunoblotted with indicated MCM2 antibodies or MCM2 antibodies that were pre-incubated with MCM2 phosphoserine-139 (pS139) peptide or serine-139 (S139) peptide.

• Figure 4 - Determination of expression, phosphorylation and chromatin association of MCM2 during the cell cycle using FACS analysis. (A) Asynchronous growing HeLa cells were fixed in ethanol and stained with propidium iodide (PI), α-MCM2M1 or α-MCM2S1 and fluorescein isothiocyanate (FITC)-labeled secondary antibodies. The upper panel shows the expression and phosphorylation levels of MCM2 determined by α-MCM2M1 staining (in blue) and α-MCM2S1 (in red) in cells at different stages of the cell cycle. The lower panel represents the cell cycle profiles (PI staining). (B) Asynchronous growing HeLa cells were extracted with CSK buffer prior to ethanol fixation. The fixed cells were then stained with propidium iodide (PI), α-MCM2M1 or α-MCM2S1 and fluorescein isothiocyanate (FITC)-labeled secondary antibodies as in (A). The upper panel shows chromatin-bound MCM2 and its phosphorylation levels determined by α-MCM2M1 staining (in blue) and α-MCM2S1 (in red) in cells at different stages of the cell cycle. The lower panel represents the cell cycle profiles (PI staining). Note: In the bivariate plots comparing PI and FITC staining, FITC as measured in the FL1 detector (515-545nm) increases in a manner dependent upon PI fluorescence. This effect, referred to as "spectral overlap", can be attributed to the brightness and broad emission spectrum of PI. Although PI has an emission peak around 615nm, when excited by a 488nm laser, some of the emitted photons will have shorter wavelengths, even down into the green range of the visible light spectrum. This is very common and to be expected.

• Figure 5 - Purification of MCM2-7, MCM2A-7 or MCM2E-7 complex by Superose-6 gel filtration column. MCM2-7, MCM2A-7 or MCM2E-7 complex was expressed by baculovirus system and was purified by anti-FLAG antibody affinity column as described under “Materials and Methods”. The affinity-purified MCM2-7 (A), MCM2A-7 (B) or MCM2E-7 (C) complex was loaded to Superose-6 gel filtration column and fractionated through AKTA-FPLC system. The fraction 23 and 24 were collected, concentrated, and used for the other experiments. Arrows indicate the positions of marker proteins (Thy: thyroglobulin, IgG: immunoglobulin, Ova: Ovalbumin) and asters indicate the fraction 23 and 24.