Abstract
Using a bio-oligo pull-down DNA-binding assay we investigated the binding capacity of endogenous, DNA damage-induced p53 in human diploid fibroblasts to several p53-responsive elements (REs) present in p53-regulated genes. During the course of p53 accumulation, we observed a decrease in p53 binding to the GADD45 but not to the p21(WAF1/CIP1) RE. Using mutated GADD45 sequences we show that this change is dependent on the presence of cytosines at position 3 in RE pentamers and on the p53 redox state. Site-directed mutagenesis experiments demonstrated that Cys277 (a residue directly contacting base 3 in a RE pentamer) is critical for differential regulation of GADD45 in DNA-damaged cells. These data represent a novel mechanism for differential affinity of p53 to distinct REs.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Substitution / genetics
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Cell Line
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins / genetics
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Cysteine / chemistry
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Cysteine / metabolism*
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DNA / chemistry
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DNA / genetics
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DNA / metabolism*
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DNA Damage*
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Fibroblasts
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GADD45 Proteins
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Humans
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Intracellular Signaling Peptides and Proteins
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Kinetics
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Mutation / genetics
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Oxidation-Reduction
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Protein Binding / radiation effects
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Proteins / genetics
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Radiation, Ionizing
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Response Elements / genetics*
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Substrate Specificity
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Thermodynamics
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Thymine / metabolism
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Time Factors
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Tumor Suppressor Protein p53 / chemistry*
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Tumor Suppressor Protein p53 / genetics
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Tumor Suppressor Protein p53 / metabolism*
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Ultraviolet Rays
Substances
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CDKN1A protein, human
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Cyclin-Dependent Kinase Inhibitor p21
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Cyclins
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Intracellular Signaling Peptides and Proteins
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Proteins
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Tumor Suppressor Protein p53
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DNA
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Cysteine
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Thymine