The interaction of SV40 large T antigen with unspecific double-stranded DNA: an electron microscopic study.
T antigen, an early protein encoded by simian virus 40 (SV40), is a specific DNA-binding protein with high affinity for elements in the viral origin of replication where it forms a double-hexameric complex as a prerequisite for DNA untwisting and, in the presence of ATP hydrolysis, for DNA unwinding. Like other specific DNA-binding proteins, T antigen also associates with DNA strands of random sequence albeit at reduced affinity. In addition, T antigen is able to unwind unspecific DNA sequences starting from internal binding sites. This property could be a step in the pathway leading to the chromosomal rearrangements that are frequently observed in SV40-transformed cells. This possibility prompted us to investigate the binding of T antigen to unspecific DNA using electron microscopy. We observed that the protein binds randomly to many unspecific DNA sites excluding a preference for particular DNA sequences or structural features. Addition of ATP to the binding buffer induces the formation of oligomeric, possibly hexameric, T antigen complexes that frequently align to form long arrays of DNA-bound protein. Magnesium salts induce the formation of tightly packed T antigen aggregates which bind to DNA to form many DNA branches and loops that emanate from the aggregated protein core. Upon ATP hydrolysis, aggregated T antigen catalyzes the unwinding of DNA duplices.
Wessel
R
R
Division of Biology, Universität Konstanz, Germany.
Ramsperger
U
U
Stahl
H
H
Knippers
R
R
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000952:Antigens, Polyomavirus Transforming / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D001665:Binding Sites; D004279:DNA, Viral / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D016591:Histocytological Preparation Techniques; D008854:Microscopy, Electron; D012680:Sensitivity and Specificity
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate