Association of Human Purα with the Retinoblastoma Protein, Rb, Regulates Binding to the Single-stranded DNA Purα Recognition Element

Abstract

The retinoblastoma protein, Rb, is detected in extracts of monkey CV-1 cells complexed with Purα, 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 Purα or Rb. The Purα•Rb complexes contain a form of Purα with extensive post-synthetic modification, as demonstrated following expression of Purα cDNA fused to a 9-amino acid epitope tag. Human Purα, 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-Purα binds to p56RB, an NH2-terminal-truncated Rb protein purified from Escherichia coli, containing the T-antigen binding domain, to form multimeric complexes. The single-stranded DNA Purα recognition element disrupts these complexes. Conversely, high concentrations of p56RB prevent Purα binding to DNA. Through use of a series of deletion mutants, the DNA binding activity of Purα is localized to a series of modular amino acid repeats. Rb binding involves a Purα region with limited homology to the Rb-binding region of SV40 large T-antigen. Binding of Purα to p56RB, the COOH-terminal portion of Rb, is inhibited by a synthetic peptide containing the T-antigen Rb-binding motif. The retinoblastoma protein, Rb, is detected in extracts of monkey CV-1 cells complexed with Purα, 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 Purα or Rb. The Purα•Rb complexes contain a form of Purα with extensive post-synthetic modification, as demonstrated following expression of Purα cDNA fused to a 9-amino acid epitope tag. Human Purα, 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-Purα binds to p56RB, an NH2-terminal-truncated Rb protein purified from Escherichia coli, containing the T-antigen binding domain, to form multimeric complexes. The single-stranded DNA Purα recognition element disrupts these complexes. Conversely, high concentrations of p56RB prevent Purα binding to DNA. Through use of a series of deletion mutants, the DNA binding activity of Purα is localized to a series of modular amino acid repeats. Rb binding involves a Purα region with limited homology to the Rb-binding region of SV40 large T-antigen. Binding of Purα to p56RB, the COOH-terminal portion of Rb, is inhibited by a synthetic peptide containing the T-antigen Rb-binding motif.