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Oxidation of p53 through DNA Charge Transport Involves a Network of Disulfides within the DNA-Binding Domain

Schaefer, Kathryn N. and Geil, Wendy M. and Sweredoski, Michael J. and Moradian, Annie and Hess, Sonja and Barton, Jacqueline K. (2015) Oxidation of p53 through DNA Charge Transport Involves a Network of Disulfides within the DNA-Binding Domain. Biochemistry, 54 (3). pp. 932-941. ISSN 0006-2960. PMCID PMC4310631. doi:10.1021/bi501424v.

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Transcription factor p53 plays a critical role in the cellular response to stress stimuli. We have seen that p53 dissociates selectively from various promoter sites as a result of oxidation at long-range through DNA-mediated charge transport (CT). Here, we examine this chemical oxidation and determine the residues in p53 that are essential for oxidative dissociation, focusing on the network of cysteine residues adjacent to the DNA-binding site. Of the eight mutants studied, only the C275S mutation shows decreased affinity for the Gadd45 promoter site. However, both mutations C275S and C277S result in substantial attenuation of oxidative dissociation, with C275S causing the most severe attenuation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide-labeled, whereas oxidized cysteines participating in disulfide bonds were ^(13)C_2D_2-iodoacetamide-labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed by mass spectrometry. A distinct shift in peptide labeling toward ^(13)C_2D_2-iodoacetamide-labeled cysteines is observed in oxidized samples, confirming that chemical oxidation of p53 occurs at long range. All observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds among the cysteine network. On the basis of these data, it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

Item Type:Article
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URLURL TypeDescription Information CentralArticle
Sweredoski, Michael J.0000-0003-0878-3831
Moradian, Annie0000-0002-0407-2031
Hess, Sonja0000-0002-5904-9816
Barton, Jacqueline K.0000-0001-9883-1600
Additional Information:© 2015 American Chemical Society. This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: November 17, 2014; Revised: December 19, 2014; Publication Date (Web): January 13, 2015. This research was funded by the Ellison Foundation (AG-SS-2079-08), the Moore Foundation for support of the Center for Chemical Signaling at Caltech, the Gordon and Betty Moore Foundation (GBMF775), The Beckman Institute, and National Institutes of Health (1S10OD010788-01A1). We thank Lisa Beckmann for assistance with molecular cloning and protein purification.
Funding AgencyGrant Number
Ellison FoundationAG-SS-2079-08
Gordon and Betty Moore FoundationGBMF775
Caltech Beckman InstituteUNSPECIFIED
Issue or Number:3
PubMed Central ID:PMC4310631
Record Number:CaltechAUTHORS:20150120-083007297
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Official Citation:Oxidation of p53 through DNA Charge Transport Involves a Network of Disulfides within the DNA-Binding Domain Kathryn N. Schaefer, Wendy M. Geil, Michael J. Sweredoski, Annie Moradian, Sonja Hess, and Jacqueline K. Barton Biochemistry 2015 54 (3), 932-941 DOI: 10.1021/bi501424v
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:53855
Deposited By: Tony Diaz
Deposited On:20 Jan 2015 16:40
Last Modified:10 Nov 2021 20:08

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