Redox state of tumor suppressor p53 regulates its sequence-specific DNA binding in DNA-damaged cells by cysteine 277.
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.
Buzek
Jiri
J
Department of Virology, Haartman Institute, and Molecular Cancer Biology Program, Biomedicum Helsinki, University of Helsinki and Helsinki University Central Hospital Laboratory Diagnostics, PO Box 63, FIN-00014 Helsinki, Finland.
Latonen
Leena
L
Kurki
Sari
S
Peltonen
Karita
K
Laiho
Marikki
M
D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D005347:Fibroblasts; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000528:radiation effects; D011506:Proteins / Q000235:genetics; D011839:Radiation, Ionizing; D020218:Response Elements / Q000235:genetics*; D013379:Substrate Specificity; D013816:Thermodynamics; D013941:Thymine / Q000378:metabolism; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays; D000097642:GADD45 Proteins
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D003545:Cysteine; D004247:DNA; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D013941:Thymine; D016159:Tumor Suppressor Protein p53; D000097642:GADD45 Proteins; C493956:CDKN1A protein, human