Eukaryotic chromosomal DNA is licensed for replication precisely once in each cell cycle. The mini-chromosome maintenance (MCM) complex plays a role in this replication licensing. We have determined the structure of a fragment of MCM from Methanobacterium thermoautotrophicum (mtMCM), a model system for eukaryotic MCM. The structure reveals a novel dodecameric architecture with a remarkably long central channel. The channel surface has an unusually high positive charge and binds DNA. We also show that the structure of the N-terminal fragment is conserved for all MCMs proteins despite highly divergent sequences, suggesting a common architecture for a similar task: gripping/remodeling DNA and regulating MCM activity. An mtMCM mutant protein equivalent to a yeast MCM5 (CDC46) protein with the bob1 mutation at its N terminus has only subtle structural changes, suggesting a Cdc7-bypass mechanism by Bob1 in yeast. Yeast bypass experiments using MCM5 mutant proteins support the hypothesis for the bypass mechanism.
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Protein Data Bank
*Note: In the version of this article initially published online, this paper contained two mistakes. The first mistake is in the legend of Fig. 3b; the correct legend should read: "b, Side view of the N-mtMCM dodecamer showing the predominantly negatively charged (red) outer surface". The second mistake is in the first paragraph of page 5 (third line from the top); the correct sentence should read: "A surface charge calculation shows that the inner surface of the entire channel is strongly positive (Fig. 3c); in contrast, the outside surface is mostly negative (Fig. 3b)". This mistake has been corrected in the HTML and print versions of the article.
Diffley, J.F. & Cocker, J.H. Protein–DNA interactions at a yeast replication origin. Nature 357, 169–172 (1992).
Walter, J. & Newport, J.W. Regulation of replicon size in Xenopus egg extracts. Science 275, 993–995 (1997).
Aparicio, O.M., Weinstein, D.M. & Bell, S.P. Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91, 59–69 (1997).
Chong, J.P., Mahbubani, H.M., Khoo, C.Y. & Blow, J.J. Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature 375, 418–421 (1995).
Kubota, Y., Mimura, S., Nishimoto, S., Takisawa, H. & Nojima, H. Identification of the yeast MCM3-related protein as a component of Xenopus DNA replication licensing factor. Cell 81, 601–609 (1995).
Maiorano, D., Moreau, J. & Mechali, M. XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Nature 404, 622–625 (2000).
Tanaka, T., Knapp, D. & Nasmyth, K. Loading of an Mcm protein onto DNA replication origins is regulated by Cdc6p and CDKs. Cell 90, 649–660 (1997).
Yan, H., Merchant, A.M. & Tye, B.K. Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. Genes Dev. 7, 2149–2160 (1993).
Ishimi, Y. A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex. J. Biol. Chem. 272, 24508–24513 (1997).
Labib, K., Tercero, J.A. & Diffley, J.F. Uninterrupted MCM2-7 function required for DNA replication fork progression. Science 288, 1643–1647 (2000).
Lee, J.K. & Hurwitz, J. Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA structures. Proc. Natl. Acad. Sci. USA 98, 54–59 (2001).
Lei, M. & Tye, B.K. Initiating DNA synthesis: from recruiting to activating the MCM complex. J. Cell. Sci. 114, 1447–1454 (2001).
Labib, K. & Diffley, J.F. Is the MCM2-7 complex the eukaryotic DNA replication fork helicase? Curr. Opin. Genet. Dev. 11, 64–70 (2001).
Tye, B.K. MCM proteins in DNA replication. Annu. Rev. Biochem. 68, 649–686 (1999).
Patel, S.S. & Picha, K.M. Structure and function of hexameric helicases. Annu. Rev. Biochem. 69, 651–697 (2000).
Schwacha, A. & Bell, S.P. Interactions between two catalytically distinct MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication. Mol. Cell 8, 1093–1104 (2001).
Kelman, Z., Lee, J.K. & Hurwitz, J. The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum ΔH contains DNA helicase activity. Proc. Natl. Acad. Sci. USA 96, 14783–14788 (1999).
Shechter, D.F., Ying, C.Y. & Gautier, J. The intrinsic DNA helicase activity of Methanobacterium thermoautotrophicum ΔH minichromosome maintenance protein. J. Biol. Chem. 275, 15049–15059 (2000).
Chong, J.P., Hayashi, M.K., Simon, M.N., Xu, R.M. & Stillman, B. A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase. Proc. Natl. Acad. Sci. USA 97, 1530–1535 (2000).
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).
Grabowski, B. & Kelman, Z. Autophosphorylation of archaeal Cdc6 homologues is regulated by DNA. J. Bacteriol. 183, 5459–5464 (2001).
Simmons, D.T. SV40 large T antigen functions in DNA replication and transformation. Adv. Virus. Res. 55, 75–134 (2000).
Singleton, M.R., Sawaya, M.R., Ellenberger, T. & Wigley, D.B. Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101, 589–600 (2000).
Kearsey, S.E. & Labib, K. MCM proteins: evolution, properties, and role in DNA replication. Biochim. Biophys. Acta 1398, 113–136 (1998).
Hardy, C.F., Dryga, O., Seematter, S., Pahl, P.M. & Sclafani, R.A. mcm5/cdc46-bob1 bypasses the requirement for the S phase activator Cdc7p. Proc. Natl. Acad. Sci. USA 94, 3151–3155 (1997).
Valle, M., Gruss, C., Halmer, L., Carazo, J.M. & Donate, L.E. Large T-antigen double hexamers imaged at the simian virus 40 origin of replication. Mol. Cell. Biol. 20, 34–41 (2000).
Fanning, E. & Knippers, R. Structure and function of simian virus 40 large tumor antigen. Annu. Rev. Biochem. 61, 55–85 (1992).
Edwards, M.C. et al. MCM2-7 complexes bind chromatin in a distributed pattern surrounding ORC in Xenopus egg extracts. J. Biol. Chem. 277, 33049–33057 (2002).
Bochkarev, A., Bochkareva, E., Frappier, L. & Edwards, A.M. The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for single-stranded DNA binding. EMBO J. 18, 4498–4504 (1999).
Poplawski, A., Grabowski, B., Long, S.E. & Kelman, Z. The zinc finger domain of the archaeal minichromosome maintenance protein is required for helicase activity. J. Biol. Chem. 276, 49371–49377 (2001).
Yu, X. et al. The Methanobacterium thermoautotrophicum MCM protein can form heptameric rings. EMBO Rep. 3, 792–797 (2002).
Smelkova, N.V. & Borowiec, J.A. Dimerization of simian virus 40 T antigen hexamers activates T-antigen DNA helicase activity. J. Virol. 71, 8766–8773 (1997).
Gulbis, J.M., Kelman, Z., Hurwitz, J., O'Donnell, M. & Kuriyan, J. Structure of the C-terminal region of p21(WAF1/CIP1) complexed with human PCNA. Cell 87, 297–306 (1996).
Mauguen, Y. et al. Molecular structure of a new family of ribonucleases. Nature 297, 162–164 (1982).
Spiller, B., Gershenson, A., Arnold, F.H. & Stevens, R.C. A structural view of evolutionary divergence. Proc. Natl. Acad. Sci. USA 96, 12305–12310 (1999).
Le Du, M.H., Stigbrand, T., Taussig, M.J., Menez, A. & Stura, E.A. Crystal structure of alkaline phosphatase from human placenta at 1.8 Å resolution. Implication for a substrate specificity. J. Biol. Chem. 276, 9158–9165 (2001).
Dalton, S. & Hopwood, B. Characterization of Cdc47p-minichromosome maintenance complexes in Saccharomyces cerevisiae: identification of Cdc45p as a subunit. Mol. Cell. Biol. 17, 5867–5875 (1997).
Lei, M. et al. Mcm2 is a target of regulation by Cdc7-Dbf4 during the initiation of DNA synthesis. Genes Dev. 11, 3365–3374 (1997).
Jones, T.A., Zou, J.Y. & Cowen, S.W. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1990).
Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).
Aiyar, A., Xiang, Y. & Leis, J. Site-directed mutagenesis using overlap extension PCR. Methods Mol. Biol. 57, 177–191 (1996).
Rothstein, R. Disruption, replacement and allele rescue: integrative DNA transformation in yeast. in Guide to Yeast Genetics and Molecular Biology (ed. Guthrie, C. & Fink, G.) 281–301 (Academic Press, San Diego; 1991).
Sclafani, R.A., Tecklenburg, M. & Pierce, A. The mcm5-bob1 bypass of Cdc7p/Dbf4p in DNA replication depends on both Cdk1-independent and Cdk1-dependent steps in Saccharomyces cerevisiae. Genetics 161, 47–57 (2002).
Kraulis, P.J. MOLSCRIPT — a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).
Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).
We thank S. Harrison for comments on the manuscript, L. Pessoa-Brandão, R. Zhao, D. Li, T. Gould and L. Wilson for assistance and other members of the Chen group for comments and input; R. Zhang at 19id in Argonne National Laboratory (APS) and the staff at 14bmc in APS and X25 and X4A in Brookhaven National Laboratory for assistance in data collection; and the UCHSC X-ray center in Biomolecular Structure Program for support. This work is supported by start-up and cancer-center funds from UCHSC to X.C. and an NIH grant to R.S.
The authors declare no competing financial interests.
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Modulation of Gene Silencing by Cdc7p via H4 K16 Acetylation and Phosphorylation of Chromatin Assembly Factor CAF-1 in Saccharomyces cerevisiae
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