The replication of SV40 DNA has been studied by many investigators as a paradigm for the replication of eukaryotic and more specifically mammalian DNA. Although the molecules involved in the synthesis of SV40 DNA are known and many of their functions elucidated, there are serious gaps in our knowledge of the mechanism of DNA replication. In particular, we understand little of the way in which initiation and elongation complexes assemble on SV40 DNA and of the protein-protein and protein-DNA interactions that are needed for efficient DNA synthesis. There is only rudimentary understanding of the timing of various events during DNA replication. Our state of knowledge therefore allows only a sketchy picture of the mechanics of DNA synthesis. To obtain this information, we have developed 3 specific aims. 1. To study the requirements for assembly of SV40 large T antigen into functional double hexamers on the origin.
This aim will be carried out primarily by studying mutants of T antigen that are defective in assembly. 2. To elucidate the mechanism of origin unwinding and timing of events that occur when the origin is unwound by the T antigen initiation machine. Various approaches will be taken to deduce how this works. Our goal here is to deduce the changes that take place to the DNA and to T antigen during unwinding 3. To characterize the assembly of initiation and elongation complexes so as to better understand the details of DNA replication. We will study the order in which complexes assemble and how the various components work together to carry out DNA replication. It is anticipated that a much better understanding of the mechanism of SV40 DNA replication will spill over to the characterization of mammalian DNA replication and provide the basic knowledge that is required to target replication factors and develop adequate therapies to treat human diseases such as cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA036118-22
Application #
7414827
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Blair, Donald G
Project Start
1985-09-01
Project End
2010-04-30
Budget Start
2008-05-01
Budget End
2010-04-30
Support Year
22
Fiscal Year
2008
Total Cost
$290,619
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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