A new DNA binding assay to study subclasses of SV40 large T antigen binding to individual binding sites on the SV40 DNA.

SV40 large T antigen interacts specifically with three different sequences, termed sites I, II or III, within the control region of the SV40 DNA to regulate transcription as well as the initiation and progress of SV40 DNA replication. We have biotinylated three different DNAs containing either site I, II or III and immobilized these constructs on a streptavidin agarose matrix. All three immobilized DNAs were shown to bind T antigen from a total cell extract of SV40 infected monkey cells although with different affinities. Pulse chase experiments revealed that newly synthesized T antigen bound efficiently to all three binding sites whereas mature T antigen bound only to site I and site III DNA. The analysis of non-binding and DNA-bound T antigen on sucrose density gradients showed that only low molecular weight forms of T antigen were bound to all three immobilized DNAs. However, incubation of T antigen in the total cell extract with site I DNA resulted in high molecular weight forms of T antigen, indicating that the presence of site I DNA influences the quaternary structure of T antigen which might result in a detachment from the DNA.