Abstract
Homologous recombination is repressed by the binding of p53 to Rad51. We identified by fluorescence and NMR spectroscopy that peptides corresponding to residues 179-190 of Rad51 bind to the core domain of p53 in a site that overlaps with its specific DNA binding site. The p53 site is quite promiscuous, since it also binds peptides derived from 53BP1, 53BP2, Hif-1alpha, and BCL-X(L) in overlapping regions. Binding is mediated mainly by a strong, nonspecific, electrostatic component and is fine tuned by specific interactions. Competition of the different proteins with each other and with specific DNA for a single site in p53 could be a factor in regulation of its activity.
Highlights
The tumor suppressor protein p53 is a transcription factor that maintains genome integrity by two means: transcriptional pathways, which trigger processes that lead to cell cycle arrest or apoptosis in response to oncogenic stress [1, 2], and nontranscriptional processes, which include binding Rad51 and thereby inhibiting homologous recombination [3]. p53 is a homotetramer that consists of several structural and functional domains
We identified by fluorescence and NMR spectroscopy that peptides corresponding to residues 179 –190 of Rad51 bind to the core domain of p53 in a site that overlaps with its specific DNA binding site
Our findings indicate that the DNA-binding interface of p53CD is a promiscuous site for protein binding, which functions via a large electrostatic component that is fine tuned by specific interactions
Summary
Peptide Synthesis—Peptides were synthesized on a Pioneer peptide synthesizer (Applied Biosystems) using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. The fluorescein-labeled peptides were dissolved in 50 mM Hepes buffer, pH 7.2, 5 mM dithiothreitol at the desired ionic strength to a final concentration of 0.05– 0.1 M. Data were fit to a simple 1:1 equilibrium model: R ϭ R0 ϩ (⌬R*[P]/ ([P] ϩ Kd), where R represents the measured fluorescence anisotropy value, ⌬R is amplitude of the fluorescence change from the initial value (peptide only) to the final value (peptide in complex), [P] is the added protein concentration, R0 is the starting fluorescence value, corresponding to the free peptide, and Kd is the dissociation constant for the complex. The ionic strength was adjusted using NaCl. For highly charged peptides, such as poly(Glu), nonspecific binding was observed in 10 mM Hepes, pH 7.2, due to very strong electrostatic interactions. Samples for NMR experiments contained 15N-labeled wildtype or mutant p53 core domain at a concentration of 180 M and the corresponding peptides at a concentration of 1–2 mM. Chemical shift analysis was performed as described [22]
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