We have previously mapped and characterized the DNA-binding domain of the SV70 tumor (T) antigen. Our data demonstrate that a region mapping between residues 131 to 371 is important for replication origin-specific DNA binding. By protease digestion experiments, we have also shown that a """"""""core"""""""" region mapping from approximately 140 to 281 is tightly associated with the DNA. Genetic data from other labs indicate that the essential sequences within this core region map from 140 to about 260. Together, these findings define the minimal region of T antigen which is needed to bind to the replication origin. As part of this proposed work, we plan to identify the sequences within the core element that are essential for the proper structure of the DNA- binding region and for making contacts with the DNA. This will be done by testing the activity of mutants with specific amino acid substitutions. Beside DNA-binding, the SV40 T antigen has several other activities that are involved in virus DNA replication. These include an ATPase, a single-strand DNA-binding activity, a helicase and an origin-specific DNA unwinding activity. Although the origin DNA-binding and ATPase domains have been carefully mapped, there is presently very little information about the regions that are responsible for the other three activities. We plan to map these regions by examining the in vitro activity of various tryptic fragments. Finally, in order to understand how the multiple functional domains of this protein communicate and interact with one another during viral DNA replication, we will generate various deletion mutants and test the effect of the deletions on T antigen function.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA036118-05
Application #
3173625
Study Section
Virology Study Section (VR)
Project Start
1985-09-01
Project End
1993-06-30
Budget Start
1989-07-01
Budget End
1990-06-30
Support Year
5
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Delaware
Department
Type
Schools of Arts and Sciences
DUNS #
059007500
City
Newark
State
DE
Country
United States
Zip Code
19716
Mason, Aaron C; Roy, Rupa; Simmons, Daniel T et al. (2010) Functions of alternative replication protein A in initiation and elongation. Biochemistry 49:5919-28
Foster, Erin C; Simmons, Daniel T (2010) The SV40 large T-antigen origin binding domain directly participates in DNA unwinding. Biochemistry 49:2087-96
Wang, Weiping; Simmons, Daniel T (2009) Simian virus 40 large T antigen can specifically unwind the central palindrome at the origin of DNA replication. J Virol 83:3312-22
Khopde, Sujata; Simmons, Daniel T (2008) Simian virus 40 DNA replication is dependent on an interaction between topoisomerase I and the C-terminal end of T antigen. J Virol 82:1136-45
Khopde, Sujata; Roy, Rupa; Simmons, Daniel T (2008) The binding of topoisomerase I to T antigen enhances the synthesis of RNA-DNA primers during simian virus 40 DNA replication. Biochemistry 47:9653-60
Wang, Weiping; Manna, David; Simmons, Daniel T (2007) Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication. J Virol 81:4510-9
Simmons, Daniel T; Gai, Dahai; Parsons, Rebekah et al. (2004) Assembly of the replication initiation complex on SV40 origin DNA. Nucleic Acids Res 32:1103-12
Roy, Rupa; Trowbridge, Pamela; Yang, Zheng et al. (2003) The cap region of topoisomerase I binds to sites near both ends of simian virus 40 T antigen. J Virol 77:9809-16
Jiao, Junfang; Simmons, Daniel T (2003) Nonspecific double-stranded DNA binding activity of simian virus 40 large T antigen is involved in melting and unwinding of the origin. J Virol 77:12720-8
Wu, C; Roy, R; Simmons, D T (2001) Role of single-stranded DNA binding activity of T antigen in simian virus 40 DNA replication. J Virol 75:2839-47

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