Structural and kinetic analysis of p53-DNA complexes and comparison of human and murine p53.
Sequence-specific DNA binding by p53 is dependent upon protein conformation. The 1620+ form correlates with wild type p53 suppressor function and is a prerequisite for binding to the DNA consensus p53-CON in vitro. It has been reported that murine p53 changes conformation on interaction with high affinity DNA target sequences and in the present study we have analysed p53-DNA complexes using conformation-specific monoclonal antibodies against p53. For murine p53 (mp53) we show (i) the 1620+ form is retained and stabilised in complex with DNA, and (ii) the complexes are dissociated by the PAb1620 monoclonal antibody. In contrast, PAb1620 did not detect nor dissociate human p53-DNA complexes nor did it interfere with complex formation. In competition experiments murine p53 replaced human p53 (hp53) in p53-DNA complexes and this correlated with the greater lability observed for hp53-DNA complexes at a given temperature. Mixed human-murine p53 oligomers were competent for DNA binding with an estimated affinity around 5 x 10(-10) M, similar to that observed for either human or murine p53 alone. The potential significance of these observations is discussed in relation p53 function in vivo.
Hall
A R
AR
Department of Biology, University of York, UK.
Milner
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013045:Species Specificity; D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D016159:Tumor Suppressor Protein p53; D004247:DNA