Abstract
Binding of Cdc6 to the origin recognition complex (ORC) is a key step in the assembly of a pre-replication complex (pre-RC) at origins of DNA replication. ORC recognizes specific origin DNA sequences in an ATP-dependent manner. Here we demonstrate cooperative binding of Saccharomyces cerevisiae Cdc6 to ORC on DNA in an ATP-dependent manner, which induces a change in the pattern of origin binding that requires the Orc1 ATPase. The reaction is blocked by specific origin mutations that do not interfere with the interaction between ORC and DNA. Single-particle reconstruction of electron microscopic images shows that the ORC–Cdc6 complex forms a ring-shaped structure with dimensions similar to those of the ring-shaped MCM helicase. The ORC-Cdc6 structure is predicted to contain six AAA+ subunits, analogous to other ATP-dependent protein machines. We suggest that Cdc6 and origin DNA activate a molecular switch in ORC that contributes to pre-RC assembly.
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References
Bell, S.P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002).
Stillman, B. Origin recognition and the chromosome cycle. FEBS Lett. 579, 877–884 (2005).
Kelly, T.J. & Brown, G.W. Regulation of chromosome replication. Annu. Rev. Biochem. 69, 829–880 (2000).
Bell, S.P. & Stillman, B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357, 128–134 (1992).
Rao, H. & Stillman, B. The origin recognition complex interacts with a bipartite DNA binding site within yeast replicators. Proc. Natl. Acad. Sci. USA 92, 2224–2228 (1995).
Rowley, A., Cocker, J.H., Harwood, J. & Diffley, J.F. Initiation complex assembly at budding yeast replication origins begins with the recognition of a bipartite sequence by limiting amounts of the initiator, ORC. EMBO J. 14, 2631–2641 (1995).
Makise, M. et al. Kinetics of ATP binding to the origin recognition complex of Saccharomyces cerevisiae. J. Biol. Chem. 278, 46440–46445 (2003).
Klemm, R.D. & Bell, S.P. ATP bound to the origin recognition complex is important for preRC formation. Proc. Natl. Acad. Sci. USA 98, 8361–8367 (2001).
Klemm, R.D., Austin, R.J. & Bell, S.P. Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex. Cell 88, 493–502 (1997).
Kelly, T.J. et al. The fission yeast cdc18+ gene product couples S phase to START and mitosis. Cell 74, 371–382 (1993).
Liang, C., Weinreich, M. & Stillman, B. ORC and Cdc6p interact and determine the frequency of initiation of DNA replication in the genome. Cell 81, 667–676 (1995).
Santocanale, C. & Diffley, J.F. ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae. EMBO J. 15, 6671–6679 (1996).
Cocker, J.H., Piatti, S., Santocanale, C., Nasmyth, K. & Diffley, J.F. An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast. Nature 379, 180–182 (1996).
Donovan, S., Harwood, J., Drury, L.S. & Diffley, J.F. Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc. Natl. Acad. Sci. USA 94, 5611–5616 (1997).
Perkins, G. & Diffley, J.F. Nucleotide-dependent prereplicative complex assembly by Cdc6p, a homolog of eukaryotic and prokaryotic clamp-loaders. Mol. Cell 2, 23–32 (1998).
Weinreich, M., Liang, C. & Stillman, B. The Cdc6p nucleotide-binding motif is required for loading mcm proteins onto chromatin. Proc. Natl. Acad. Sci. USA 96, 441–446 (1999).
Wang, B. et al. The essential role of Saccharomyces cerevisiae CDC6 nucleotide-binding site in cell growth, DNA synthesis, and Orc1 association. J. Biol. Chem. 274, 8291–8298 (1999).
Mizushima, T., Takahashi, N. & Stillman, B. Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro. Genes Dev. 14, 1631–1641 (2000).
Seki, T. & Diffley, J.F. Stepwise assembly of initiation proteins at budding yeast replication origins in vitro. Proc. Natl. Acad. Sci. USA 97, 14115–14120 (2000).
Feng, L., Hu, Y., Wang, B., Wu, L. & Jong, A. Loss control of Mcm5 interaction with chromatin in cdc6–1 mutated in CDC-NTP motif. DNA Cell Biol. 19, 447–457 (2000).
Liang, C. & Stillman, B. Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants. Genes Dev. 11, 3375–3386 (1997).
Marahrens, Y. & Stillman, B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science 255, 817–823 (1992).
Wilmes, G.M. & Bell, S.P. The B2 element of the Saccharomyces cerevisiae ARS1 origin of replication requires specific sequences to facilitate pre-RC formation. Proc. Natl. Acad. Sci. USA 99, 101–106 (2002).
Zou, L. & Stillman, B. Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase. Mol. Cell. Biol. 20, 3086–3096 (2000).
Diffley, J.F., Cocker, J.H., Dowell, S.J., Harwood, J. & Rowley, A. Stepwise assembly of initiation complexes at budding yeast replication origins during the cell cycle. J. Cell Sci. 19 (suppl.), 67–72 (1995).
Feng, L., Wang, B., Driscoll, B. & Jong, A. Identification and characterization of Saccharomyces cerevisiae Cdc6 DNA-binding properties. Mol. Biol. Cell 11, 1673–1685 (2000).
Takenaka, H. et al. ADP-binding to origin recognition complex of Saccharomyces cerevisiae. J. Mol. Biol. 340, 29–37 (2004).
Takahashi, N. et al. Analysis on origin recognition complex containing Orc5p with defective Walker A motif. J. Biol. Chem. 279, 8469–8477 (2004).
Schepers, A. & Diffley, J.F. Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein. J. Mol. Biol. 308, 597–608 (2001).
Bell, S.P., Mitchell, J., Leber, J., Kobayashi, R. & Stillman, B. The multidomain structure of Orc1p reveals similarity to regulators of DNA replication and transcriptional silencing. Cell 83, 563–568 (1995).
Bowers, J.L., Randell, J.C., Chen, S. & Bell, S.P. ATP hydrolysis by ORC catalyzes reiterative Mcm2–7 assembly at a defined origin of replication. Mol. Cell 16, 967–978 (2004).
Fletcher, R.J. et al. The structure and function of MCM from archaeal M. thermoautotrophicum. Nat. Struct. Biol. 10, 160–167 (2003).
Bowman, G.D., Goedken, E.R., Kazmirski, S.L., O'Donnell, M. & Kuriyan, J. DNA polymerase clamp loaders and DNA recognition. FEBS Lett. 579, 863–867 (2005).
Tugal, T. et al. The Orc4p and Orc5p subunits of the Xenopus and human origin recognition complex are related to Orc1p and Cdc6p. J. Biol. Chem. 273, 32421–32429 (1998).
Giraldo, R. & Diaz-Orejas, R. Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p). Proc. Natl. Acad. Sci. USA 98, 4938–4943 (2001).
Giraldo, R. Common domains in the initiators of DNA replication in Bacteria, Archaea and Eukarya: combined structural, functional and phylogenetic perspectives. FEMS Microbiol. Rev. 26, 533–554 (2003).
Gajiwala, K.S. & Burley, S.K. Winged helix proteins. Curr. Opin. Struct. Biol. 10, 110–116 (2000).
Liu, J. et al. Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control. Mol. Cell 6, 637–648 (2000).
Blow, J.J. & Hodgson, B. Replication licensing–defining the proliferative state? Trends Cell Biol. 12, 72–78 (2002).
Lee, D.G., Makhov, A.M., Klemm, R.D., Griffith, J.D. & Bell, S.P. Regulation of origin recognition complex conformation and ATPase activity: differential effects of single-stranded and double-stranded DNA binding. EMBO J. 19, 4774–4782 (2000).
Lee, D.G. & Bell, S.P. ATPase switches controlling DNA replication initiation. Curr. Opin. Cell Biol. 12, 280–285 (2000).
Harvey, K.J. & Newport, J. Metazoan origin selection: origin recognition complex chromatin binding is regulated by CDC6 recruitment and ATP hydrolysis. J. Biol. Chem. 278, 48524–48528 (2003).
Beall, E.L. et al. Role for a Drosophila Myb-containing protein complex in site-specific DNA replication. Nature 420, 833–837 (2002).
Iyer, L.M., Leipe, D.D., Koonin, E.V. & Aravind, L. Evolutionary history and higher order classification of AAA+ ATPases. J. Struct. Biol. 146, 11–31 (2004).
Elsasser, S., Lou, F., Wang, B., Campbell, J.L. & Jong, A. Interaction between yeast Cdc6 protein and B-type cyclin/Cdc28 kinases. Mol. Biol. Cell 7, 1723–1735 (1996).
Lopez-Girona, A., Mondesert, O., Leatherwood, J. & Russell, P. Negative regulation of Cdc18 DNA replication protein by Cdc2. Mol. Biol. Cell 9, 63–73 (1998).
Weinreich, M., Liang, C., Chen, H.H. & Stillman, B. Binding of cyclin-dependent kinases to ORC and Cdc6p regulates the chromosome replication cycle. Proc. Natl. Acad. Sci. USA 98, 11211–11217 (2001).
Nguyen, V.Q., Co, C. & Li, J.J. Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature 411, 1068–1073 (2001).
Radermacher, M., Wagenknecht, T., Verschoor, A. & Frank, J. Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J. Microsc. 146, 113–136 (1987).
Karplus, K. et al. Combining local-structure, fold-recognition, and new fold methods for protein structure prediction. Proteins 53 (suppl.), 491–496 (2003).
Acknowledgements
We thank A. Stenlund for comments on the manuscript, S.P. Bell for ORC mutants and P. Wendell for technical assistance. This work was supported by a grant from the US National Institutes of Health (GM45436). H.L. acknowledges support from Brookhaven National Laboratory LDRD project number 05-112 and US Department of Energy grant KP1102010. C.S. was a fellow of the Leukemia and Lymphoma Society.
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Supplementary information
Supplementary Fig. 1
Extended footprint formation is salt sensitive at low ORC concentrations. (PDF 415 kb)
Supplementary Fig. 2
Gel-shift assay of P32 labeled ARS1 DNA with ORC and Cdc6. (PDF 83 kb)
Supplementary Fig. 3
ATP and ADP dependent footprint of ORC (PDF 225 kb)
Supplementary Fig. 4
Gel-shift assay with ORC and Cdc6 in the presence of ATP, ATPγS or ADP. (PDF 78 kb)
Supplementary Fig. 5
DNase I footprint with ORC and Cdc6 in the presence of 1 mM ADP. (PDF 374 kb)
Supplementary Fig. 6
DNase I footprint with ORC and Cdc6 with ATP and ATPγS. (PDF 632 kb)
Supplementary Fig. 7
ORC-Cdc6 interaction analyzed by glycerol gradient sedimentation in the presence of ADP. (PDF 580 kb)
Supplementary Fig. 8
Superposition of the ORC map with the ORC-Cdc6 map. (PDF 399 kb)
Supplementary Video 1
Three-dimensional structures of the yeast ORC in the presence of ATPγS, as described in Figure 4. (MOV 1138 kb)
Supplementary Video 2
Three-dimensional structures of the yeast ORC-Cdc6 complex in the presence of ATPγS. (MOV 1087 kb)
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Speck, C., Chen, Z., Li, H. et al. ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. Nat Struct Mol Biol 12, 965–971 (2005). https://doi.org/10.1038/nsmb1002
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DOI: https://doi.org/10.1038/nsmb1002
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