ASSOCIATION OF HUMAN PUR-ALPHA WITH THE RETINOBLASTOMA PROTEIN, RB, REGULATES BINDING TO THE SINGLE-STRANDED-DNA PUR-ALPHA RECOGNITION ELEMENT

The retinoblastoma protein, Rb, is detected in extracts of monkey CV-1 cells complexed with Pur alpha, a Sequence-specific single-stranded D NA-binding protein implicated in control of gene transcription and DNA replication, These complexes can be immunoextracted from cell lysates using monoclonal antibodies to either Pur alpha or Rb. The Pur alpha . Rb complexes contain a form of Pur alpha with extensive post-synthet ic modification, as demonstrated following expression of Pur alpha cDN A fused to a 9-amino acid epitope tag. Human Pur alpha, expressed as a glutathione S-transferase fusion protein, specifically binds to the h ypophosphorylated form of Rb with an affinity as high as that of SV40 large T-antigen, In the absence of DNA, glutathione S-transferase-Pur alpha binds to p56(RB), an NH2-terminal-truncated Rb protein purified from Escherichia coli, containing the T-antigen binding domain, to for m multimeric complexes, The single-stranded DNA Pur alpha recognition element disrupts these complexes, Conversely, high concentrations of p 56(RB) prevent Pur alpha binding to DNA, Through use of a series of de letion mutants, the DNA binding activity of Pur alpha is localized to a series of modular amino acid repeats, Rb binding involves a Pur alph a region with limited homology to the Rb-binding region of SV40 large T-antigen, Binding of Pur alpha to p56(RB), the COOH-terminal portion of Rb, is inhibited by a synthetic peptide containing the T-antigen Rb -binding motif.