DDK phosphorylation of Mcm2 is required for cell growth. Protein expression and purification- Mcm2: Full-length Mcm2 and fragments were

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1 DDK phosphorylation of Mcm2 is required for cell growth Supplemental Data Protein expression and purification- Mcm2: Full-length Mcm2 and fragments were cloned into pet33b (NdeI/BamHI). The plasmids were transformed into Escherichia coli Rossetta TM 2(BL21) (EMD Biosciences) and a colony from transformation was used to inoculate 6 L of ZYM-5052 auto-inducing media (Studier, 2005) containing 100 µg/ml kanamycin and 34 µg/ml chloramphenicol. The cells were grown at 37 C until an A 600 of 0.6 was reached, the temperature was then lowered to 14 C and the cells were allowed to express protein for 16 hours. Harvested cells were lysed by French press in 200 ml of 50 mm Tris-HCl ph 8.0, 500 mm NaCl, and 50 mm imidazole. The lysate was then applied to a 20 ml NiSO 4 -charged chelating Sepharose fast flow resin (GE Healthcare). The column was washed with the same buffer. The protein was then eluted in buffer 50 mm Tris-HCl ph 8.0, 100 mm NaCl, 10% glycerol, and 250 mm imidazole. Peak fractions were then dialyzed against 50 mm Tris-HCl ph 8.0, 50 mm NaCl, 10% glycerol and applied to an 8 ml SourceQ resin (GE Healthcare). The protein was then eluted with a 20-ml linear gradient from 50 mm-500 mm NaCl. Peak fractions were pooled and frozen. GST-Dbf4-Cdc7 The S. serevisiae Dbf4 gene was cloned into the NcoI/BamHI sites of pet28a (Figure 1) or pet41a vector (Figures 2-6). The Cdc7 gene was cloned into the NdeI/BamHI sites of pet16b (Figure 1) or pet11a (Figure 2-6). The expression and purification of Cdc7 alone or Dbf4 alone were essentially the same as that described

2 above for Mcm2. For the co-expression of DDK (Dbf4-Cdc7, Figures 2-6), both plasmids were transformed into Escherichia coli Rossetta TM 2(BL21) and the transformation mixture was plated on ampicillin and kanamycin. A colony from transformation was used to inoculate 6 L of ZYM-5052 auto-inducing media (Studier, 2005) containing 100 µg/ml kanamycin, 200 µg/ml ampicillin, and 34 µg/ml chloramphenicol. The DDK protein complex was then purified essentially as described above for Mcm2. Mcm2-7 reconstitution and Radiolabeling- The Mcm2-7 complex was reconstituted as previously described (Davey et al., 2003), except the Mcm3 protein contained an N- terminal site for protein kinase A phosphorylation. The Mcm2-7 complex was radiolabeled with protein kinase A in a reaction volume of 100 µl that contained 2.2 µm Mcm2-7 protein in Kinase reaction buffer (5 mm Tris-HCl ph 8.5, 10 mm MgCl 2, 1 mm

3 DTT, 500 µm ATP, 100 µci 32 P-γ-ATP) containing 5 µg PKA. Reactions were incubated at 30 C for 1 hr. Plasmids used in this study: pib201 (prs416 CEN6/ARSH4 MCM2 URA3) pib202 (prs415 CEN6/ARSH4 GAL1::MCM2 LEU2) pib203 (prs415 CEN6/ARSH4 GAL1::mcm2 S164A LEU2) pib204 (prs415 CEN6/ARSH4 GAL1::mcm2 S170A LEU2) pib205 (prs415 CEN6/ARSH4 GAL1::mcm2 S164A,S170A LEU2) Strains used in this study: Strain Genotype Source of reference BY4743 MATa/α his 3 1 leu2 0 met15 0 lys2 0 Open Biosystems ura3 0 MCM2+/mcm2::KanMX4 RSY311 MATa bar1 trp1 leu2 ura3 can1 his6 cyh2 Hoang et al. (2007) RSY727 MATa bar1 trp1 leu2 ura3 can1 his6 cyh2 Hoang et al. (2007) mcm5-bob1-1 cdc7 ::HIS3 his3-1 IRB311 RSY311; mcm2 :: KanMX4(pRS416 This study CEN6/ARSH4 MCM2+Ura3) IRB727 RSY727; mcm2 :: KanMX4(pRS416 This study CEN6/ARSH4 MCM2+Ura3) Yeast strains bearing the bob1 mutation were generously provided by Robert Sclafani (Haase and Reed, 2002). Vectors and genomic DNA were acquired from ATCC. S. cerevisiae yeast strain S288C was used as a source of genomic DNA. PCR products were cloned into XhoI /BamHI restriction sites of prs415 and prs416 vectors. Mutations and deletions were verified by PCR and DNA sequencing.

4 RSY311 and RSY 727 strains were transformed with the plasmid pib201. Overlap PCR method was used to introduce mcm2::kanmx4 deletion into haploid strains RSY311 and RSY727. The template for PCR was genomic DNA from Strain BY4743 carrying the mcm2::kanmx4 deletion (MATa/α his 3 1 leu2 0 met15 0 lys2 0 ura3 0 MCM2+/mcm2::KanMX4, Open Biosystems). The following sets of primers were used to generate two PCR products; MCM2-150upsteam-forward (5 - ATA GGA CAA AAA ATT TCG AGA AAC GAA ACC AG-3 )/KanMX4-reverse(5 -CGA CAT TAT CGC GAG CCC ATT TAT ACC CAT-3 ) and MCM2-150downstream-reverse (5 - TTT AGT TGA ATT GAA ATT AAA GAT ACA GTG TAT AA-3 )/KanMX4-up (5 -ATG GGT ATA AAT GGG CTC GCG ATA ATG TCG-3 ). The full-length, gel purified PCR product was transformed into the yeast strains. Site-specific integration into MCM2 was confirmed by the inability of the strain to survive in the absence of wild-type MCM2 (pib201) and by PCR reactions using either set of primers mentioned previously. Expression of mcm2 mutants was confirmed by RT PCR. The mcm2 mutants were tested for function and survival in vivo using a plasmid shuffle assay. The mcm2 mutants were transformed into IRB 311(mcm2 ::KanMX4 + prs416/mcm2 + ), IRB 727(mcm2 ::KanMX4 mcm5-bob1-1 cdc7 ::HIS3 + prs416/mcm2 + ), and selected on SCM-Leu-Ura plates. Transformants were re-streaked on SCM-Leu-Ura (2% Dextrose) plates. Single colonies were grown in YPD overnight, and then 10 fold serial dilutions were plated on both SCM-Leu-Ura Gal (2% Galactose) plates and SCM-Leu-FOA Gal (2% Galactose) plates. Clones that carried mutant mcm2 and were FOA-resistant were used for phenotypic studies.

5 References Davey, M. J., Indiani, C., and O'Donnell, M. (2003). Reconstitution of the Mcm2-7p heterohexamer, subunit arrangement, and ATP site architecture. J. Biol. Chem. 278, Haase, S., and Reed, S. (2002). Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle 1, Studier, F. W. (2005). Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41,

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