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.
Department of Biology, University of York, UK.
A R
AR
Hall
J
J
Milner
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