ATPase-dependent quality control of DNA replication origin licensing

Abstract

The regulated loading of the Mcm2-7 DNA helicase (comprising six related subunits, Mcm2 to Mcm7) into pre-replicative complexes at multiple replication origins ensures precise once per cell cycle replication in eukaryotic cells. The origin recognition complex (ORC), Cdc6 and Cdt1 load Mcm2-7 into a double hexamer bound around duplex DNA in an ATP-dependent reaction, but the molecular mechanism of this origin 'licensing' is still poorly understood. Here we show that both Mcm2-7 hexamers in Saccharomyces cerevisiae are recruited to origins by an essential, conserved carboxy-terminal domain of Mcm3 that interacts with and stimulates the ATPase activity of ORC-Cdc6. ATP hydrolysis can promote Mcm2-7 loading, but can also promote Mcm2-7 release if components are missing or if ORC has been inactivated by cyclin-dependent kinase phosphorylation. Our work provides new insights into how origins are licensed and reveals a novel ATPase-dependent mechanism contributing to precise once per cell cycle replication.

Figure 1. Mcm3 is necessary and sufficient for Mcm2-7 recruitment

a) Protein requirements for Mcm2-7 recruitment. ORC, Cdc6, Cdt1 and Mcm2-7 were purified as described in Supplementary Methods and in Supp. Fig. 1. 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 Supplementary Methods and bound proteins were analysed by immunoblot with the indicated antibodies. b) Recruitment of Mcm2-7 by ORC and Cdc6 was performed as in Fig. 1a. 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).

Figure 2. The C-terminus of Mcm3 is required for Mcm2-7 recruitment

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.

Figure 3. Both Mcm2-7 hexamers must interact with ORC•Cdc6 through Mcm3

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.

Figure 4. Mcm3 binding activates the ORC•Cdc6 ATPase

The conversion of α³²P-ATP to α³²P-ADP was monitored as described in Supplementary Methods 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.

Figure 5. ATP hydrolysis by ORC•Cdc6 can promote Mcm2-7 release

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 Supplementary Methods, 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.