a) Protein requirements for Mcm2-7 recruitment. ORC, Cdc6, Cdt1 and Mcm2-7 were purified as described in and in . After incubation of the indicated proteins with DNA beads in the presence of ATPγS, beads were isolated and washed with low salt. DNA was uncoupled with irradiation as described in and bound proteins were analysed by immunoblot with the indicated antibodies. b) Recruitment of Mcm2-7 by ORC and Cdc6 was performed as in . Reactions contained Mcm2-7 either with (+) or without (−) Cdt1. Bound proteins were visualised by silver staining (i) or immunoblot (ii). Panel (iii) summarises the result in Fig. 1b (i and ii).

a) The domain architecture of Mcm3 and alignment of the Mcm3 C-terminus. The alignment includes Mcm3 from a variety of eukaryotic species (Sc, Saccharomyces cerevisiae; Kl, Kluyveromyces lactis; Yl, Yarrowia lipolytica; Sp, Schizosaccharomyces pombe; Hs, Homo sapiens; Xl, Xenopus laevis; Dm, Drosophila melanogaster). Residue numbers above alignment correspond to S. cerevisiae Mcm3. The position of various mutants is shown by vertical lines, and the mutant amino acid residue is shown at the bottom of the line. Allele names are on the right. The numbering of alleles begins with 11 to prevent confusion with any existing mcm3 mutant alleles. b) Recruitment assay performed with a C-terminal MBP tagged Mcm3 fragment (last 194 aa) and with the indicated proteins. c) Cdt1•Mcm2-7 complexes containing wild type and mutant Mcm3 were tested for recruitment or d) for loading, proteins were analysed by silver staining. e) Wild type and mutant Mcm3 alleles were expressed from a galactose-inducible promoter in strains containing the mcm3-td degron mutant. A dilution series was tested for growth on plates containing either 2% D-glucose or 2% D-galactose at either 25°C or 37°C as indicated.

a) Complexes containing wild type 3x FLAG Mcm3 and MBP Mcm3-11 were assembled (IN) and mixed in the indicated ratios, with a fixed amount of the wild type complex (4 pmol) and tested for loading and recruitment as above. b) Loading and recruitment of mixed complexes containing 3x FLAG Mcm3-11 and MBP Mcm3 wt in the indicated ratios.

The conversion of α³²P-ATP to α³²P-ADP was monitored as described in in reactions containing the indicated proteins (Mcm3 wild type; Mcm3-13 mutant; Mcm3-CT, a polypeptide containing the C-terminal 194 amino acid residues of Mcm3; ORC and Cdc6). Error bars depict standard error of the mean from five reactions.

a) Mcm3 was tested for recruitment (low salt wash) in reactions containing ORC, Cdc6 and either ATP or ATPγS. Bound proteins were analysed by silver staining (silver) or immunoblotting with anti-Mcm3 antibody (immuno). b) Recruitment and loading assays were performed as above with ORC, Cdc6 and Mcm2-7 either with (+) or without (-) Cdt1 in ATP or ATPγS. Proteins were visualized by silver staining. c) Complexes containing either the complete six subunit ORC complex (All) or a complex lacking Orc6 (Δ6), purified as described in , were tested for recruitment (low salt wash) in either ATP or ATPγS. DNA bound proteins were analysed by silver staining. d) Recruitment and loading assays were performed with unphosphorylated ORC (ORC) or CDK-phosphorylated ORC (ORC-P). In lanes 9-12 ORC-P was previously dephosphorylated with lambda phosphatase. e) A revised model for the mechanism of origin licensing which includes an ATPase dependent release step based on experiments in this manuscript. Details of the model are discussed in the text. The “?” in step (v) refers to the assembly of the second hexamer, which is discussed in the text.