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RESEARCH
[ CNB ]

[ CSIC ]
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MCM, the study of Archaeal helicases

The MCM complex is currently considered the best candidate for the eukaryotic replicative helicase. All six components of the eukaryotic MCM heterohexamer (Forsburg, 2004) are conserved from yeast to human, contain an MCM-specific version of the ATPase Walker A and B motifs, and are essential for DNA synthesis initiation and elongation. However, its mode of action is not clear, since helicase activity has only been found for an Mcm4,6,7 subcomplex and not for the complete purified MCM hexamer. The great number of additional proteins involved in coordinating eukaryotic DNA replication with the cell cycle further complicates the problem.

Genome sequencing has shown that the DNA replication machinery is highly similar in Archaea and Eukarya (Whitman et al., 1999, Cann and Ishino, 1999). In collaboration with Dr. Xiaogianj S. Chen (University of Southern California), we study the archaeal helicase MCM from Methanobacterium thermoautotrophicum (mtMCM) to understand helicase function and DNA replication initiation in archaea and, by extension, in eukaryotic cells. M. thermoautotrophicum has a single MCM homolog (Smith et al., 1997), which has clearly been shown to function as a helicase (Kelman et al., 1999, Chong et al., 2000, Shechter et al., 2000). The crystal structure of the mtMCM N-terminal domain showed a cylindrical structure formed by two hexameric rings bound head to head, and traversed by a positively charged channel (Fletcher et al., 2003). However, EM studies of the full length protein have failed to provide a 3D image of the double hexamer structure. Instead, single 6-fold, and surprisingly, 7-fold ring architectures, have been reported (Yu et al., 2002, Pape et al., 2003), as well as helical arrangements (Chen et al., 2005).

We use 3DEM to image the structure of mtMCM in conditions as close as possible to its functional state. Combination of 3DEM medium resolution maps with high resolution data of mtMCM or other homolog proteins will help to elucidate the mode of action of MCM helicases during replication initiation and elongation.

References

  • Cann, I. K. and Ishino, Y. (1999) Genetics, 152, 1249-67. [ PubMed abstract ]
  • Chen, Y.-J., Yu, X., Kasiviswanathan, R., Shin, J.-H., Kelman, Z. and Egelman, E. H. (2005) J Mol Biol, in press. [ PubMed abstract ]
  • Chong, J. P., Hayashi, M. K., Simon, M. N., Xu, R. M. and Stillman, B. (2000) Proc Natl Acad Sci USA, 97, 1530-5. [ PubMed abstract ]
  • Fletcher, R. J., Bishop, B. E., Leon, R. P., Sclafani, R. A., Ogata, C. M. and Chen, X. S. (2003) Nat Struct Biol, 10, 160-7. [ PubMed abstract ]
  • Forsburg, S. L. (2004) Microbiol Mol Biol Rev, 68, 109-31, table of contents. [ PubMed abstract ]
  • Kelman, Z., Lee, J. K. and Hurwitz, J. (1999) Proc Natl Acad Sci USA, 96, 14783-8. [ PubMed abstract
  • Pape, T., Meka, H., Chen, S., Vicentini, G., Van Heel, M. and Onesti, S. (2003) EMBO Rep, 4, 1079-83. [ PubMed abstract ]
  • Shechter, D. F., Ying, C. Y. and Gautier, J. (2000) J Biol Chem, 275, 15049-59. [ PubMed abstract ]
  • Smith, D. R., Doucette-Stamm, L. A., Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P., Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R., Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D., Reeve, J. N. and et al. (1997) J Bacteriol, 179, 7135-55. [ PubMed abstract ]
  • Whitman, W. B., Pfeifer, F., Blum, P. and Klein, A. (1999) Genetics, 152, 1245-8. [ PubMed abstract ]
  • Yu, X., VanLoock, M. S., Poplawski, A., Kelman, Z., Xiang, T., Tye, B. K. and Egelman, E. H. (2002) EMBO Rep, 3, 792-7. [ PubMed abstract ]