Although we have learned a great deal about the proteins involved in eukaryotic DNA replication, we know relatively little about how they communicate with one another to initiate DNA synthesis and to carry out the elongation reaction. Nearly everything we know about this process in high eukaryotes has come from the study of SV40. This virus codes for only one protein directly involved in DNA synthesis; the remaining factors come from the cell. T antigen associates with a number of these proteins, including DNA polymerase alpha, RPA and topoisomerase I (topoI) and there is accumulating evidence that the interaction of T antigen with these cellular proteins is required for the replication of SV40 DNA. We have concentrated on the association between T antigen and topoI and have obtained evidence that these two proteins may form the first functional complex at the origin of replication. Our goal is to build on these observations to obtain a clearer picture of the order in which cellular proteins associated with one another to form the initiation complex and to determine the function of each protein during initiation and elongation. In particular, we would like to better understand the relationship between the T antigen helicase and topoisomerase to better appreciate how these two enzymes communicate with one another during DNA unwinding. Since this complex must deal with the single strands that are generated during unwinding, we would also like to characterize the single-stranded DNA binding domain of T antigen which we have recently shown to be completely separate from the origin- binding domain. We therefore want to analyze the role of topoisomerase I in SV40 DNA replication, study the composition of the complex(es) that participates in SV40 DNA replication and determine the function of each protein in the complex, and characterize the function of the single- stranded DNA binding domain of T antigen in an effort to gain insights into the mechanism of helicase action.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA036118-17
Application #
6628231
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
1985-09-01
Project End
2004-06-30
Budget Start
2003-02-01
Budget End
2004-06-30
Support Year
17
Fiscal Year
2003
Total Cost
$295,674
Indirect Cost
Name
University of Delaware
Department
Biology
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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