title: Redox state regulates binding of p53 to sequence-specific DNA, but not to non-specific or mismatched DNA.
abstract: Redox modulation of wild-type p53 plays a role in sequence-specific DNA binding in vitro . Reduction produces a DNA-binding form of the protein while oxidation produces a non-DNA-binding form. Primer extension analysis reveals that increasing concentrations of reduced p53 result in enhanced protection of the consensus sequence, while increasing concentrations of oxidized p53 confer minimal protection of the consensus sequence. DNA binding by oxidized p53 is, therefore, not sequence-specific. In contrast, there is no observable difference in the binding of oxidized p53 and reduced p53 to double-stranded non-specific or mismatched DNA in gel mobility shift assays. Both forms of p53 bind equally well, suggesting that redox modulation of p53 does not play a role in its binding to non-specific or mismatched DNA. In view of the in vitro evidence that redox state influences the sequence-specific DNA-binding of p53, we have examined the effect of oxidative stress on the in vivo ability of p53 to bind to and transactivate PG13-CAT, a reporter construct containing multiple copies of the p53 consensus binding site linked to the chloramphenicol acetyltransferase gene. Hydrogen peroxide treatment of cells cotransfected with p53 results in a marked decrease in CAT activity, suggesting that oxidation of p53 decreases the ability of the protein to bind to consensus DNA and transactivate target genes in vivo.
authors.affiliation: Biology Department, University of Alaska, 3211 Providence Drive, Anchorage, AK 99508, USA.
authors.forename: D
authors.initials: D
authors.lastname: Parks
authors.forename: R
authors.initials: R
authors.lastname: Bolinger
authors.forename: K
authors.initials: K
authors.lastname: Mann
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I; D017930:Genes, Reporter; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051379:Mice; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA; D006861:Hydrogen Peroxide; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I