The retinoblastoma protein, Rb, is detected in extracts of monkey CV-1 cells complexed with Pur, a sequence-specific single-stranded DNA-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 or Rb. The PurRb complexes contain a form of Pur with extensive post-synthetic modification, as demonstrated following expression of Pur cDNA fused to a 9-amino acid epitope tag. Human Pur, expressed as a glutathione S-transferase fusion protein, specifically binds to the hypophosphorylated form of Rb with an affinity as high as that of SV40 large T-antigen. In the absence of DNA, glutathione S-transferase-Pur binds to p56, an NH-terminal-truncated Rb protein purified from Escherichia coli, containing the T-antigen binding domain, to form multimeric complexes. The single-stranded DNA Pur recognition element disrupts these complexes. Conversely, high concentrations of p56 prevent Pur binding to DNA. Through use of a series of deletion mutants, the DNA binding activity of Pur is localized to a series of modular amino acid repeats. Rb binding involves a Pur region with limited homology to the Rb-binding region of SV40 large T-antigen. Binding of Pur to p56, the COOH-terminal portion of Rb, is inhibited by a synthetic peptide containing the T-antigen Rb-binding motif.

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