Many DNA tumor viruses encode """"""""initiators"""""""" proteins that site specifically bind to viral origins of DNA replication. Upon binding to their respective origins, the initiators assemble into DNA helicases that are capable of melting duplex DNA. Initiators also recruit cellular proteins required for synthesis of nascent DNA. We are attempting to understand these processes by characterizing in depth an initiator encoded by Simian Virus 40, termed T-antigen (T-ag). Using the Simian Virus 40 system and simple band shift experiments, a fundamental question in biology will be addressed: """"""""how does the cell cycle machinery control the assembly of initiators on viral origins of replication?"""""""" Recent experiments with T-ag derived peptides, whose ability to bind to DNA is regulated by phosphorylation of Thr 124, are providing significant insights into this process. Experiments are proposed to determine if similar mechanisms are operating in the context of T-ag. Related aspects of T-ag assembly and regulation will be considered, such as locating a site on T-ag whose interactions with the flanking sequences in the core origin is necessary for T-ag binding. Collectively, these studies will provide information that may allow us to solve the mechanism of DNA unwinding. Once the SV40 origin is unwound, the pol alpha-primase complex initiates the synthesis of nascent DNA strands. Exactly what sequences constitute the initiation sites for the pol alpha-primase complex is the topic of an additional series of experiments. Given that many basic processes are conserved throughout evolution, we are confident that the information we obtain from these studies will be pertinent to the initiation of DNA replication at other viral origins of replication. Furthermore, they may help us to understand how the cell cycle machinery controls initiation events at cellular origins. Given that uncontrolled DNA replication is thought to be a component of many diseases, including cancer, it is very important to understand how DNA replication is initiated and how it is controlled.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055397-13
Application #
6844339
Study Section
Virology Study Section (VR)
Program Officer
Rhoades, Marcus M
Project Start
1992-01-01
Project End
2006-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
13
Fiscal Year
2005
Total Cost
$309,075
Indirect Cost
Name
Tufts University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
039318308
City
Boston
State
MA
Country
United States
Zip Code
02111
Meinke, Gretchen; Phelan, Paul J; Harrison, Celia J et al. (2013) Analysis of the costructure of the simian virus 40 T-antigen origin binding domain with site I reveals a correlation between GAGGC spacing and spiral assembly. J Virol 87:2923-34
Harrison, Celia J; Meinke, Gretchen; Kwun, Hyun Jin et al. (2011) Asymmetric assembly of Merkel cell polyomavirus large T-antigen origin binding domains at the viral origin. J Mol Biol 409:529-42
Meinke, Gretchen; Phelan, Paul J; Fradet-Turcotte, Amélie et al. (2011) Structure-based analysis of the interaction between the simian virus 40 T-antigen origin binding domain and single-stranded DNA. J Virol 85:818-27
Meinke, Gretchen; Phelan, Paul; Fradet-Turcotte, Amélie et al. (2011) Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain. Acta Crystallogr D Biol Crystallogr 67:560-7
Fradet-Turcotte, Amelie; Morin, Genevieve; Lehoux, Michael et al. (2010) Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication. Virology 399:65-76
Kumar, Anuradha; Joo, Woo S; Meinke, Gretchen et al. (2008) Evidence for a structural relationship between BRCT domains and the helicase domains of the replication initiators encoded by the Polyomaviridae and Papillomaviridae families of DNA tumor viruses. J Virol 82:8849-62
Meinke, Gretchen; Phelan, Paul; Moine, Stephanie et al. (2007) The crystal structure of the SV40 T-antigen origin binding domain in complex with DNA. PLoS Biol 5:e23
Kumar, Anuradha; Meinke, Gretchen; Reese, Danielle K et al. (2007) Model for T-antigen-dependent melting of the simian virus 40 core origin based on studies of the interaction of the beta-hairpin with DNA. J Virol 81:4808-18
Fradet-Turcotte, Amelie; Vincent, Caroline; Joubert, Simon et al. (2007) Quantitative analysis of the binding of simian virus 40 large T antigen to DNA. J Virol 81:9162-74
Reese, Danielle K; Meinke, Gretchen; Kumar, Anuradha et al. (2006) Analyses of the interaction between the origin binding domain from simian virus 40 T antigen and single-stranded DNA provide insights into DNA unwinding and initiation of DNA replication. J Virol 80:12248-59

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