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Structural and kinetic analysis of p53-DNA complexes and comparison of human and murine p53
Abstract:
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.
Insights
The p53 protein
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- p53 protein conformation is critical for sequence-specific DNA binding.
- The 1620+ conformation of p53 is linked to wild-type suppressor function and DNA binding.
- Murine p53 undergoes conformational changes upon binding to DNA.
Purpose of the Study:
- To investigate p53-DNA complex formation and stability using conformation-specific antibodies.
- To compare the DNA binding characteristics of murine p53 (mp53) and human p53 (hp53).
Main Methods:
- Utilized conformation-specific monoclonal antibodies (e.g., PAb1620) to analyze p53-DNA complexes.
- Performed competition experiments with mp53 and hp53 for DNA binding.
- Assessed the lability of p53-DNA complexes at varying temperatures.
Main Results:
- Murine p53 (mp53) retains and stabilizes the 1620+ conformation when bound to DNA.
- The PAb1620 antibody dissociates mp53-DNA complexes but not hp53-DNA complexes.
- hp53-DNA complexes exhibit greater lability than mp53-DNA complexes.
- Mixed human-murine p53 oligomers bind DNA with an affinity similar to homooligomers.
Conclusions:
- Conformation-specific antibodies can differentiate between murine and human p53-DNA interactions.
- Differences in complex stability may influence p53 function in vivo.
- The study provides insights into the regulation of p53 DNA binding and its implications for tumor suppression.