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Title: The Crystal Structure of the SV40 T-Antigen Origin Binding Domain in Complex with DNA.

Abstract

No abstract prepared.

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930272
Report Number(s):
BNL-80971-2008-JA
TRN: US200822%%1439
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: PLoS Biology; Journal Volume: 5; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; CRYSTAL STRUCTURE; DNA; ORIGIN; national synchrotron light source

Citation Formats

Meinke,G., Phelan, P., Moine, S., Bochkareva, E., Bochkarev, A., Bullock, P., and Bohm, A.. The Crystal Structure of the SV40 T-Antigen Origin Binding Domain in Complex with DNA.. United States: N. p., 2007. Web. doi:10.1371/journal.pbio.0050023.
Meinke,G., Phelan, P., Moine, S., Bochkareva, E., Bochkarev, A., Bullock, P., & Bohm, A.. The Crystal Structure of the SV40 T-Antigen Origin Binding Domain in Complex with DNA.. United States. doi:10.1371/journal.pbio.0050023.
Meinke,G., Phelan, P., Moine, S., Bochkareva, E., Bochkarev, A., Bullock, P., and Bohm, A.. Mon . "The Crystal Structure of the SV40 T-Antigen Origin Binding Domain in Complex with DNA.". United States. doi:10.1371/journal.pbio.0050023.
@article{osti_930272,
title = {The Crystal Structure of the SV40 T-Antigen Origin Binding Domain in Complex with DNA.},
author = {Meinke,G. and Phelan, P. and Moine, S. and Bochkareva, E. and Bochkarev, A. and Bullock, P. and Bohm, A.},
abstractNote = {No abstract prepared.},
doi = {10.1371/journal.pbio.0050023},
journal = {PLoS Biology},
number = 2,
volume = 5,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. Tomore » overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS-PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 {angstrom} resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner.« less
  • With the aim of forming the ‘lock-washer’ conformation of the origin-binding domain of SV40 large T antigen in solution, using structure-based analysis an intermolecular disulfide bridge was engineered into the origin-binding domain to generate higher order oligomers in solution. The 1.7 Å resolution structure shows that the mutant forms a spiral in the crystal and has the de novo disulfide bond at the protein interface, although structural rearrangements at the interface are observed relative to the wild type. The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of themore » viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner.« less
  • The origin-binding domain (OBD) of simian virus 40 (SV40) large T-antigen (T-Ag) is essential for many of T-Ag's interactions with DNA. Nevertheless, many important issues related to DNA binding, for example, how single-stranded DNA (ssDNA) transits along the T-Ag OBD, have yet to be established. Therefore, X-ray crystallography was used to determine the costructure of the T-Ag OBD bound to DNA substrates such as the single-stranded region of a forked oligonucleotide. A second structure of the T-Ag OBD crystallized in the presence of poly(dT){sub 12} is also reported. To test the conclusions derived from these structures, residues identified as beingmore » involved in binding to ssDNA by crystallography or by an earlier nuclear magnetic resonance study were mutated, and their binding to DNA was characterized via fluorescence anisotropy. In addition, these mutations were introduced into full-length T-Ag, and these mutants were tested for their ability to support replication. When considered in terms of additional homology-based sequence alignments, our studies refine our understanding of how the T-Ag OBDs encoded by the polyomavirus family interact with ssDNA, a critical step during the initiation of DNA replication.« less
  • The origins of replication of DNA tumor viruses have a highly conserved feature, namely, multiple binding sites for their respective initiator proteins arranged as inverted repeats. In the 1.45- Angstroms crystal structure of the simian virus 40 large T-antigen (T-ag) origin-binding domain (obd) reported herein, T-ag obd monomers form a left-handed spiral with an inner channel of 30 Angstroms having six monomers per turn. The inner surface of the spiral is positively charged and includes residues known to bind DNA. Residues implicated in hexamerization of full-length T-ag are located at the interface between adjacent T-ag obd monomers. These data providemore » a high-resolution model of the hexamer of origin-binding domains observed in electron microscopy studies and allow the obd's to be oriented relative to the hexamer of T-ag helicase domains to which they are connected.« less