Abstract
The p53 gene encodes a nuclear phosphoprotein that is biologically activated in response to genotoxic stresses including treatment with anticancer platinum drugs. The DNA binding activity of p53 protein is crucial for its tumor suppressor function. DNA interactions of active wild-type human p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by antitumor cisplatin and its clinically ineffective trans isomer (transplatin) were investigated by using a gel mobility shift assay. It was found that DNA adducts of cisplatin reduced binding affinity of the consensus DNA sequence to p53, whereas transplatin adducts did not. This result was interpreted to mean that the precise steric fit required for the formation and stability of the tetrameric complex of p53 with the consensus sequence cannot be attained, as a consequence of severe conformational perturbations induced in DNA by cisplatin adducts. The results also demonstrate an increase of the binding affinity of p53 to DNA lacking the consensus sequence and modified by cisplatin but not by transplatin. In addition, only major 1,2-GG intrastrand cross-links of cisplatin are responsible for this enhanced binding affinity of p53. The data base on structures of various DNA adducts of cisplatin and transplatin reveals distinctive structural features of 1,2-intrastrand cross-links of cisplatin, suggesting a unique role for this adduct in the binding of p53 to DNA lacking the consensus sequence. The results support the hypothesis that the mechanism of antitumor activity of cisplatin may also be associated with its efficiency to affect the binding affinity of platinated DNA to active p53 protein.
Highlights
It is well established that platinum coordination complexes exhibit antitumor effects [1, 2]
The 474-bp fragment was further globally modified by cisplatin or transplatin at rb ϭ 0.02– 0.1, and the unplatinated 2513-bp fragment was added as the nonspecific competitor
This result demonstrates the inefficiency of transplatin adducts to reduce the binding affinity of the consensus DNA response element (CDRE) to the active p53 protein
Summary
It is well established that platinum coordination complexes exhibit antitumor effects [1, 2]. We have examined binding of DNA lacking CDRE and modified by cisplatin or transplatin to reveal affinity of active wt p53 to platinated DNA containing no consensus nucleotide sequence.
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