title: Dimerization of simian virus 40 T-antigen hexamers activates T-antigen DNA helicase activity.
abstract: Chromosomal DNA replication in higher eukaryotes takes place in DNA synthesis factories containing numerous replication forks. We explored the role of replication fork aggregation in vitro, using as a model the simian virus 40 (SV40) large tumor antigen (T antigen), essential for its DNA helicase and origin-binding activities. Previous studies have shown that T antigen binds model DNA replication forks primarily as a hexamer (TAgH) and to a lesser extent as a double hexamer (TAgDH). We find that DNA unwinding in the presence of ATP or other nucleotides strongly correlates with the formation of TAgDH-DNA fork complexes. TAgH- and TAgDH-fork complexes were isolated, and the TAgDH-bound fork was denatured at a 15-fold-higher rate during the initial times of unwinding. TAgDH bound preferentially to a DNA substrate containing a 50-nucleotide bubble, indicating the bridging of each single-stranded DNA/duplex DNA junction, and this DNA molecule was also unwound at a high rate. Both the TAgH- and TAgDH-fork complexes were relatively stable, with the half-life of the TAgDH-fork complex greater than 40 min. Our data therefore indicate that the linking of two viral replication forks serves to activate DNA replication.
authors.affiliation: Department of Biochemistry and Kaplan Comprehensive Cancer Center, New York University Medical Center, New York 10016, USA.
authors.forename: N V
authors.initials: NV
authors.lastname: Smelkova
authors.forename: J A
authors.initials: JA
authors.lastname: Borowiec
mesh_terms: D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry* / Q000378:metabolism; D002474:Cell-Free System; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D019281:Dimerization; D007700:Kinetics; D011994:Recombinant Proteins; D013539:Simian virus 40 / Q000201:enzymology / Q000235:genetics*; D013329:Structure-Activity Relationship
chemical_list: D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D011994:Recombinant Proteins; D004265:DNA Helicases