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RESEARCH
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The large T antigen of Simian Virus 40

Simian Virus 40 (SV40), a polyomavirus that produces different diseases in monkeys, induces tumours in rodents and inmortalization in several cultured mammalian cell lines, has been used as a simplified model to the understanding of the eukaryotic replication process (Simmons, 2000). The origin of replication (ori) for SV40 genome, unlike that of the eukaryotic cells, it is very well characterized and is specifically recognized by a multi-functional viral protein, the large T antigen (LTag), a hexameric replicative helicase. In the presence of Mg2+ and ATP LTag forms a double hexamer complex covering the complete ori core, and thus, LTag can be envisaged as a functional homolog of eukaryotic MCM protein (minichromosome maintenance factor), since both unwind duplex DNA and assemble as double hexamers (Fletcher, 2003).

At the initial steps of the replication LTag binds to specific sequences at the SV40 ori. The core of this DNA region has a length of 64 bp and displays three domains (Deb et al., 1986): a central region called site II of about 27 base pairs in length, harbouring a perfect palindrome that includes four GAGGC pentanucleotides which are the specific binding sites for LTag, and another two flanking regions, an AT-rich domain upstream the LTag binding sites and an imperfect inverted repeat, termed early palindrome (EP), downstream the central region (Fanning and Knippers, 1992). When LTag recognises this DNA sequence, the protein multimerises into some sort of bilobed structure that was further described as a double hexamer (Dean et al., 1987). The assembly of the LTag double hexamer requires ATP (Borowiec and Hurwitz, 1988) and has to proceed through the binding to a "head-to-head" oriented pair derived from the four available pentanucleotides at the core origin of replication (Joo et al., 1998).

Our group has focused on the structural characterization of LTag double hexamers assembled on the SV40 ori using electron microscopy and image processing techniques. Initial 2D structural studies (Valle et al., 2000) showed important features of this nucleoprotein complex, such as its overall shape and size, and provided the first mapping of its functional domains. Clearly, the LTag-ori complex was shown as a bilobed structure made up by two hexamers arranged in a head-to-head orientation. The assignment of the N-terminal and C-terminal regions, based on monoclonal antibody labeling (Valle et al., 2000) allowed us to describe the current model.

Later on, the first 3D structure of the full-length Tag double hexamer was determined using cryo-electron microscopy (cryo-EM) and single-particle reconstruction techniques (Gomez-Lorenzo et al., 2003). The 3D map showed an extremely flexible double hexamer structure, with a prominent curvature along the DNA axis. The map was used to fit the atomic coordinates of Tag helicase domain (Li et al., 2003).

References

  • Borowiec, J.A. and Hurwitz, J. (1988) ATP stimulates the binding of simian virus 40 (SV40) large tumor antigen to the SV40 origin of replication. Proc Natl Acad Sci USA, 85, 64-68. [ PubMed abstract ]
  • Dean, F.B., Dodson, M., Echols, H. and Hurwitz, J. (1987) ATP-dependent formation of a specialized nucleoprotein structure by simian virus 40 (SV40) large tumor antigen at the SV40 replication origin. Proc Natl Acad Sci USA, 84, 8981-8985. [ PubMed abstract ]
  • Deb, S., DeLucia, A.L., Baur, C.P., Koff, A. and Tegtmeyer, P. (1986) Domain structure of the simian virus 40 core origin of replication. Mol Cell Biol, 6, 1663-1670. [ PubMed abstract ]
  • Fanning, E. and Knippers, R. (1992) Structure and function of simian virus 40 large tumor antigen. Annu Rev Biochem, 61, 55-85.[ PubMed abstract ]
  • Fletcher, R.J., Bishop, B.E., Leon, R.P., Sclafani, R.A., Ogata, C.M. and Chen, X.S. (2003) The structure and function of MCM from archaeal M. Thermoautotrophicum. Nat. Struct. Biol., 10, 160-167.[ PubMed abstract ]
  • Gomez-Lorenzo, M.G., Valle, M., Frank, J., Gruss, C., Sorzano, C.O., Chen, X.S., Donate, L.E. and Carazo, J.M. (2003) Large T antigen on the simian virus 40 origin of replication: a 3D snapshot prior to DNA replication. Embo J, 22, 6205-6213.[ PubMed abstract ]
  • Joo, W.S., Kim, H.Y., Purviance, J.D., Sreekumar, K.R. and Bullock, P.A. (1998) Assembly of T-antigen double hexamers on the simian virus 40 core origin requires only a subset of the available binding sites. Mol Cell Biol, 18, 2677-2687.[ PubMed abstract ]
  • Li, D., Zhao, R., Lilyestrom, W., Gai, D., Zhang, R., DeCaprio, J.A., Fanning, E., Jochimiak, A., Szakonyi, G. and Chen, X.S. (2003) Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen. Nature, 423, 512-518.[ PubMed abstract ]
  • Simmons, D.T. (2000) SV40 large T antigen functions in DNA replication and transformation. Adv Virus Res, 55, 75-134.[ PubMed abstract ]
  • Valle, M., Gruss, C., Halmer, L., Carazo, J.M. and Donate, L.E. (2000) Large T-antigen double hexamers imaged at the simian virus 40 origin of replication. Mol Cell Biol, 20, 34-41.[ PubMed abstract ]