DNA Binding Properties of Simian Virus 40 Temperature-sensitive A Proteins

Wild-type simian virus 40 A protein (large T antigen) bound to three tandem regions of simian virus 40 DNA. The binding regions were defined by the ability of A protein to protect simian virus 40 DNA from digestion with limited (footprint assay) or excess (fragment assay) amounts of DNase I. At low concentrations, protein first bound to region I, which maps 30 to 45 base pairs to the early side of the origin of replication. At higher concentrations, A protein also protected region II and then region III. Region II spanned approximately 65 base pairs and corresponded in location to the functional origin of replication that contains a unique BglI site along with an adjacent adenine-thymine-rich region. Region III was adjacent to the late boundary of region II, but its distal limit was not well defined. Twelve distinct temperature-sensitive (ts) A proteins were purified and examined for their ability to bind in regions I to III. Three classes of tsA protein were defined on the basis of thermal stability. Class I tsA protein displayed wild-type binding either with or without a heat shock. Unheated class II tsA protein exhibited wild-type binding, but after a heat shock bound very poorly to the origin of replication. Class III tsA protein was defective in its binding even without a heat shock and only protected region I. Classes II and III were coded by mutants mapping in two distinct regions of the genome. For all of the tsA proteins examined, there was a positive correlation between the thermolability of origin binding in vitro and the temperature sensitivity of these mutants for DNA replication and transcriptional autoregulation in vivo. This correlation adds support to the essential role of origin binding by A protein in viral DNA replication and early transcription repression.