Dan Simmons' Molecular Virology Lab
Our laboratory is focused on understanding the mechanism by which SV40 T antigen and various cellular proteins participate in virus DNA replication.
Classes: B401 Molecular Biology of the
Cell
B280 Fundamentals of
Biotechnology
B619 Gene Expression
Laboratory
B679 Virology
RESEARCH: Our laboratory is interested in the structure and function of the simian virus 40 tumor antigen (T antigen) and of cellular proteins that interact with it in virus infected cells. T antigen is a multifunctional phosphoprotein synthesized early in SV40 infection. It is required for virus DNA replication and for the regulation of viral gene expression in infected cells. Its main functions are to serve as the origin recognition protein and helicase so that replication can initiate at the origin and proceed bidirectionally along the circular DNA genome.
By using a multifaceted biochemical and genetic approach, we are investigating the fine structure and activity of various functional domains of T antigen and correlating this information to the biology of SV40. In addition, we are investigating the role of various cellular proteins in the initiation and elongation of DNA synthesis from the viral origin. First, we have obtained detailed information about how T antigen binds to and unwinds the origin of replication and how it participates in the initiation and elongation phases of SV40 DNA replication. This viral protein forms a double hexamer over the origin and this structure serves as the helicase that structurally distorts then melts and unwinds the origin. It also functions to separate the DNA strands at replication forks. We have obtained evidence that cellular proteins topoisomerase I (topo I), DNA polymerase α/primase (pol/prim) and replication protein A (RPA) form a complex with hexamers of T antigen to initiate DNA replication. This complex is stabilized by numerous protein-protein and protein-DNA interactions and we are in the process of mapping and characterizing as many of these as possible. We have recently obtained detailed information about how topoisomerase I interacts with T antigen. By studying the effects of various proteins on the assembly of the complex, we have developed a model that describes the order of events at the origin to initiate DNA replication. We are presently testing various aspects of this model and asking how the complex changes during various stages of DNA replication.
MODEL OF ASSEMBLY OF
REPLICATION COMPLEX ON SV40 DNA
RELEVANT PUBLICATIONS:
Simmons, D. T., T. Melendy, D. Usher and B.
Stillman. Simian Virus 40 large T antigen binds to
Chen, L., W. S. Joo, P. A. Bullock, and D. T. Simmons. The N-terminal side of the origin-binding domain of simian virus 40 large T antigen is involved in A/T untwisting, J. Virol., 71, 8743-8749, 1997. Go to Abstract
Simmons, D. T., P. W. Trowbridge, and R. Roy. Topoisomerase I stimulates SV40 T antigen-mediated DNA replication and inhibits T antigen's ability to unwind DNA at non-origin sites, Virology, 242, 435-443, 1998. Go to Abstract
Pommier, Y., Kohlagen, G. Wu, C. and D. T. Simmons. Mammalian DNA topoisomerase I activity and poisoning by camptothecin are inhibited by simian virus 40 large T antigen, Biochemistry, 37, 3813-3823, 1998. Go to paper
Simmons, D. T., Roy, R., Chen, L., and P. W. Trowbridge. The activity of topoisomerase I is modulated by large T antigen during unwinding of the SV40 origin, J. Biol.Chem., 273:20390-20396, 1998. Go to paper
Wu, C., Edgil, D. and D. T. Simmons. The Origin DNA-Binding and Single-Stranded DNA-Binding Domains of Simian Virus 40 Large T Antigen are Distinct, J. Virol., 72:10256-10259, 1998. Go to paper.
Weisshart, K., Taneja, P., Jenne, A. Herbig, U., Simmons, D. T. and Fanning, E. Two regions of SV40 T antigen determine cooperativity of double hexamer assembly on the viral origin of DNA replication and promote hexamer interactions during bidirectional origin DNA unwinding, J. Virol., 73:2201-2211, 1999. Go to paper .
Trowbridge, P. W., Roy, R. and Simmons, D. T. Human topoisomerase I promotes initiation of SV40 DNA replication in vitro. Mol. Cell. Biol. 19:1686-1694, 1999. Go to paper.
Gai, D, Roy, R. Wu, C, and Simmons, D. T. Topoisomerase I Associates Specifically with Simian Virus 40 Large T Antigen Double Hexamer-Origin Complexes, J. Virol. , 74:5224-5232, 2000. Go to paper
Simmons, D. T. SV40 Large T antigen- Functions in
DNA Replication and Transformation, Advances in Virus Research, 55:75-134,
2000.
Wu, C., R. Roy, and Simmons, D. T. Role of Single-Stranded DNA Binding Activity of T Antigen in Simian Virus 40 DNA Replication. J. Virol. 75:2839-2847, 2001. Go to paper
Prabhu, V. P., Simons, A. M., Iwasaki, H., Gai, D., Simmons, D. T. and Chen, J. p53 Blocks RuvAB Promoted Branch Migration and Modulates Resolution of Holliday Junctions by RuvC. J Mol Biol 316:1023-1032, 2002. Go to paper
Jiao, J., and Simmons, D. T. Nonspecific DNA Binding Activity of Simian virus 40 large T Antigen is Involved in Melting and Unwinding of the Origin. J. Virol., 77:12720-12728, 2003. Go to paper
Simmons, D. T., Gai, D., Parsons, R., Debes, A., and Roy, R. Assembly of the Replication Initiation Complex on SV40 Origin DNA. Nucleic Acids Res., 32: 1103-1112, 2004. Go to paper
Wang, W., D. Manna, and Simmons,
D. T. Role of the hydrophilic channels of
simian virus 40 T-antigen helicase in DNA replication. J Virol 81:4510-4519,
2007. Go
to paper
Khopde, S., and Simmons, D. T. Simian virus 40 DNA replication is dependent on an
interaction between topoisomerase I and the C-terminal end of T antigen. J
Virol 82:1136-1145. 2008. Go to paper
Khopde,
S., R. Roy, and Simmons, D.T. The Binding of Topoisomerase I to T Antigen
Enhances the Synthesis of RNA-DNA Primers during Simian Virus 40 DNA
Replication. Biochemistry 47:
9653-9660. 2008. Go
to paper
If you are interested in learning more about our work, or if you need some material from us, email me at: dsimmons@udel.edu