Perez-Arnaiz P , Bruck I , Colbert MK , Kaplan DL .

R15 GM113167 NIGMS NIH HHS

	Figure 1. Mutations at residues belonging to Mcm10 NTD coiled-coil region disrupts Mcm10 self-association in vitro. (A) Sequence alignment of the coiled-coil region from Xenopus laevis (x), Homo sapiens (h), Mus musculus (m) and Saccharomyces cerevisiae (sc) (36). Bold letters indicate identical aminoacids. Residues 16–51 from S. cerevisiae are shown. Ile26, Leu30, Arg37 and Leu40 were targeted for site-directed mutagenesis. (A and B) Mcm10-4A is Mcm10-I26A-L30A-R37A-L40A. (B) 30 pmol of wild-type GST-Mcm10, GST-Mcm10-4A or GST tag was incubated with increasing concentrations of either radiolabeled wild-type PKA-Mcm10 or PKA-Mcm10-4A at 30°C for 10 min in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. The graph represents mean values from two independent experiments and error bars indicate the standard deviation of the mean.
	Figure 2. Mcm10-4A is defective in 80mer ssDNA binding in vitro. (A) 30 pmol of wild-type GST-Mcm10, GST-Mcm10-A4 or GST tag was incubated with increasing concentrations of radiolabeled 80mer ssDNA at 30°C for 10 min in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (B) 30 pmol of wild-type GST-Mcm10, GST-Mcm10-A4 or GST tag was incubated with increasing concentrations of radiolabeled bubble DNA at 30°C for 10 min in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (C and D) 30 pmol of wild-type GST-Mcm10, GST-Mcm10-A4 or GST tag was incubated with increasing concentrations of radiolabeled 30mer dsDNA (C) or 25mer ssDNA (D) at 30°C for 10 min in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (E) 1 nM of radiolabeled ssDNA was incubated with increasing amounts of GST-Mcm10 and GST-Mcm10-4A at 4°C for 10 min in an electrophoretic mobility shift assays. The products of the assays were analyzed by 4% native PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. Graphs from (A), (B), (C), (D), and (E) represent mean values from two independent experiments and error bars indicate the standard deviation of the mean.
	Figure 3. Mcm10-4A is able to bind and recruit Cdc45 to the Mcm2–7 complex in vitro. (A) 30 pmol of wild-type GST-Mcm10, GST-Mcm10-A4 or GST tag was incubated with increasing concentrations of radiolabeled PKA-Mcm2–7 at 30°C for 10 min in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (B) 30 pmol of GST tag or GST-Cdc45 was incubated with varying amounts of radiolabeled wild-type PKA-Mcm10 or PKA-Mcm10-4A in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (C) 30 pmol of GST-Cdc45 was incubated with 10 pmol of radiolabeled PKA-Mcm2–7 and increasing amounts of wild-type Mcm10 or Mcm10-4A in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. Graphs from (A), (B) and (C) represent mean values from two independent experiments and error bars indicate the standard deviation of the mean.
	Figure 4. Mcm10-4A is slightly defective in stimulating Mcm2 phosphorylation by DDK in vitro. (A) 30 pmol of GST-Dbf4 or GST tag was incubated with increasing concentrations of radiolabeled wild-type PKA-Mcm10 or PKA-Mcm10-4A at 30°C for 10 min in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (B) 30 pmol of GST-Cdc7 or GST tag was incubated with increasing concentrations of either radiolabeled wild-type PKA-Mcm10 or PKA-Mcm10-4A at 30°C for 10 min in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (C) 5 μg of Mcm2 was incubated with 50 ng of DDK and varying amounts of wild-type Mcm10 or Mcm10-4A in a volume of 25 μl at 30°C for 1 h. The reactions were then analyzed by western blot for expression of phospho-Mcm2. Graphs from (A) and (B) represent mean values from two independent experiments and error bars indicate the standard deviation of the mean.
	Figure 5. Mcm10-4A is slightly defective in p180 binding in vitro. (A) 30 pmol of GST-p180 or GST tag was incubated with increasing concentrations of either radiolabeled PKA-Mcm10-WT or PKA-Mcm10-4A at 30°C for 10 min in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (B) 30 pmol of GST-p180 was incubated with 10 pmoles of radiolabeled PKA-Mcm2–7 and increasing amounts of wild-type Mcm10 or Mcm10-4A in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (C) 30 pmol of GST-p180 was incubated with 10 pmol of the isolated radiolabeled PKA-Mcm2–7 subunits and increasing amounts of wild-type Mcm10 in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (D) 30 pmol of GST-p180 was incubated with 10 pmol of radiolabeled PKA-Mcm2 and increasing amounts of wild-type Mcm10 or Mcm10-4A in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (E) 30 pmol of GST-p180 was incubated with 10 pmol of radiolabeled PKA-Mcm4 and increasing amounts of radiolabeled wild-type PKA-Mcm10 or PKA-Mcm10-4A in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted. (F) 30 pmol of GST-p180 was incubated with 10 pmol of radiolabeled PKA-Mcm7 and increasing amounts of radiolabeled wild-type PKA-Mcm10 or PKA-Mcm10-4A in a GST pulldown assay. The products of the pulldown were analyzed by SDS-PAGE followed by phosphorimaging. Results from similar experiments were quantified, averaged and plotted.
	Figure 6. Expression of mcm10-4A in budding yeast results in a growth defect and reduced DDK phosphorylation of Mcm2. (A) 10-fold serial dilution analysis of budding yeast mcm10-1-aid cells expressing MCM10-WT, vector and mcm10-4A from the GAL-S plasmid inducible promoter system (pRS415). Plates were incubated for 3 days at 25°C. (B) mcm10-1-aid cells expressing MCM10-WT and mcm10-4A were grown as described in Materials and Methods. Cells were analyzed by FACS with propidium iodide staining for DNA content. (C) mcm10-1-aid cells expressing MCM10-WT and mcm10-4A were grown as described in Material and Methods. C, upper panel. Mcm2 IP samples were analyzed by western blot for expression of Mcm2. C, bottom panel. Mcm2 IP samples were analyzed by western blot for expression of phospho-Mcm2. Results from similar experiments were quantified, averaged and plotted. Graph from (C) represents mean values from two independent experiments and error bars indicate the standard deviation of the mean.
	Figure 7. The growth defect observed in cells expressing mcm10-4A is not suppressed by the presence of mcm5-bob1 mutation. (A) 10-fold serial dilution analysis of budding yeast mcm10-1-aid cells expressing MCM10-WT and mcm10-4A from the GAL-S plasmid inducible promoter system (pRS415). Plates were incubated for 3 days at 25°C. (B) similar to A, except the cells harbored the mcm5-bob1 mutation. (C) mcm10-1-aid mcm5-bob1 cells expressing MCM10-WT and mcm10-4A were grown and chromatin immunoprecipitation was performed as described in Materials and Methods. Radioactive PCR bands were quantified, averaged and plotted. (D) mcm10-1-aid mcm5-bob1cells expressing MCM10-WT and mcm10-4A were grown as described in Materials and Methods. D, left panel. Whole cell extracts were analyzed by Western blot for the expression of the indicated proteins. D, right panel. Cells were immunoprecipitated with antibodies directed against Mcm2 as described in Materials and Methods, followed by western analysis with antibodies directed against Mcm2, Mcm10, Cdc45, GINS, RPA, and Pol12. Results from different experiments were quantified, averaged and plotted. Graphs from (C) and (D) represent mean values from two independent experiments and error bars indicate the standard deviation of the mean.
	Figure 8. An intact Mcm10 coiled-coil interaction surface is important for origin melting, helicase assembly and the recruitment of Pol-α to Mcm2–7. (A) Mcm2–7 complex is loaded as a double hexamer to encircle dsDNA during late M and G1 phase. In early S phase, Mcm10 stimulates DDK phosphorylation of Mcm2. S-phase cyclin dependent kinase (CDK) phosphorylates Sld2 and Sld3 and these phosphorylated proteins bind to Dpb11 to form the ternary complex Sld3-Sld2-Dpb11 that binds to the Mcm2–7 complex. The Sld3-Sld2-Dpb11 ternary complex blocks the interaction between GINS and Mcm2–7. Lagging stand ssDNA is extruded from the central ring of Mcm2–7 complex. (B) Mcm10 oligomerization promotes Mcm10-binding to the extruded lagging ssDNA strand. (C) Sld3-Sld2-Dpb11 bind to the melted, lagging ssDNA strand, allowing GINS to bind Mcm2–7 by a passive, sequestration mechanism. (D) Mcm10 oligomerization promotes the recruitment of pol α to Mcm2–7.

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