Abstract
The retinoblastoma protein, Rb, is detected in extracts of monkey CV-1 cells complexed with Pur alpha, 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 alpha or Rb. The Pur alpha-Rb complexes contain a form of Pur alpha with extensive post-synthetic modification, as demonstrated following expression of Pur alpha cDNA fused to a 9-amino acid epitope tag. Human Pur alpha, 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 alpha 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 Pur alpha recognition element disrupts these complexes. Conversely, high concentrations of p56RB prevent Pur alpha binding to DNA. Through use of a series of deletion mutants, the DNA binding activity of Pur alpha is localized to a series of modular amino acid repeats. Rb binding involves a Pur alpha region with limited homology to the Rb-binding region of SV40 large T-antigen. Binding of Pur alpha to p56RB, the COOH-terminal portion of Rb, is inhibited by a synthetic peptide containing the T-antigen Rb-binding motif.
Highlights
The retinoblastoma protein, Rb,1 product of the rb tumor suppressor gene, is modified by phosphorylation in late G1 phase of the cell cycle [1, 2]
Hypophosphorylated Rb binds to the SV40 large T-antigen [7, 8], the adenoviral protein E1A [9], and human papilloma virus protein E7 [10], each of which plays a role in both viral gene transcription and DNA replication
E2F, which binds the same region of Rb as do the D cyclins, T-antigen, and E1A, activates many genes, including several involved in aspects of DNA replication [12,13,14,15,16]
Summary
Retinoblastoma protein; aa, amino acid; GST, glutathione S-transferase; CMV, cytomegalovirus. Pur␣ recognition elements in such a configuration are present in two 98-base pair repeats located on the late side of the central palindrome of the core origin, and these elements are essential for T-antigen-mediated replication [29] These PUR elements are necessary for helical alterations in the adjacent AT-rich region effected by T-antigen helicase activity [30]. Allowing for a 2-amino acid gap, there are 9 identities in 26 residues This homology is limited, it includes several residues conserved in initiator proteins of DNA tumor viruses, and in certain cellular proteins potentially involved in initiation, including MCM2 of yeast and BM28 of human cells [19]. Rb modulates the binding of Pur␣ to its single-stranded recognition element, and presence of this element affects the binding of Rb to Pur␣
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