Single-stranded DNA mimicry in the p53 transactivation domain interaction with replication protein A

Abstract

One of many protein-protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1-120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37-57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA͞p53 interaction can be modulated. RPA70N forms an oligonucleotide͞oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast, the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37-57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide͞oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic p53N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage.DNA binding ͉ protein-protein interaction ͉ structural analysis ͉ ssDNA mimicry U pon DNA damage, the p53 tumor suppressor is activated and orchestrates a cellular response by transcriptional regulation of genes involved in cell cycle arrest and apoptosis (1, 2). p53 protein is central to an extensive network of DNA damage sensing proteinprotein and protein-nucleic acid interactions. As yet, however, details of how this network is regulated are unclear. One component of the network is replication protein A (RPA), the major single-stranded (ss) DNA-binding protein of the eukaryotic nucleus (3-5). The interaction of p53 with RPA mediates suppression of homologous recombination (6) and modulates Werner syndrome helicase activity (7). It is also linked with DNA repair and disruption of p53 and RPA complexes after DNA damage is thought to coordinate DNA repair with the p53-dependent checkpoint control (8).Because the ability of p53 to bind specific DNA target sequences via its DNA-binding core (9) (Fig. 1,) is blocked when the protein is complexed with RPA it follows that UV-mediated disruption of the complexes is predicted to favor p53 transactivation functions (10). p53-RPA complex formation is affected by the presence of various lengths of ssDNAs, because RPA, when bound to these ssDNAs, is unable to interact with p53 (10). UV radiation of cells also reduces p53-RPA complexes by a second mechanism, because hyperphosphorylated RPA does not associate with p53 (8). Thus p53-RPA interaction is subject (i) to the presence of ssDNA molecules and also (ii) to the phosphorylation status of the RPA protein.RPA is a heterotrimer (RPA70, RPA32, and RPA14; Fig. 1B) involved in many aspects of DNA metabolism such as replication, recombination, and repair (11,12). The largest subunit, RPA70, is a tandem repeat of four oligonucleotide͞oligosaccharide-binding (OB) folds (13) comprising RPA70...

p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage

“…The key residues in human p53's DNA binding core domain (including residues 102, 103, 105, 114, 115, 122 and 126) are required for interactions with RAD51, and those in the N-terminal motif (residues 37-57) are required for interactions with RPA. These key amino acid residues are absent in the ∆133p53 protein [36,37]. This might be the reason that ∆133p53 was not co-immunoprecipitated with RAD51 and RPA in this study.…”

“…Previous studies have shown that the DNA-binding core domain (94-312) of p53 is required for p53-RAD51 interactions, and its N-terminal domain (37-57) is required for p53-RPA interactions [36,37], which suggests that ∆133p53 may not be able to form a complex with these two proteins. We performed a co-immunoprecipitation (co-IP) experiment to test this hypothesis by co-transfecting HA-RAD51 or HA-RPA2 with p53, ∆133p53 or both, into H1299 cells.…”

Sequential and Synergistic Modification of Human RPA Stimulates Chromosomal DNA Repair

“…It is unlikely that the T21A mutation creates a defect in the general conformation of the RPA2 N terminus, because this region is thought to be unstructured (16,17). It is also highly unlikely that antibody recognition of any of the RPA2 phosphoresidues is directly affected by mutation of other sites for the following reasons.…”

“…The N-terminal 33 residues of RPA2 undergo both cell cycle-and stress-dependent phosphorylation on approximately nine sites (Fig. 1A), which are thought to exist in an unstructured conformation (16,17). Ser 23 and Ser 29 are constitutively modified during mitosis by cyclin B-Cdk1 (18, 19) and have been suggested to be partially modified beginning at the G 1 /S boundary by the cyclin A-Cdk2 complex (18,20,21).…”

hSSB1 regulates both the stability and the transcriptional activity of p53

“…Given that hSSB1 regulates p53 stability and that the hSSB1 homolog RPA has been shown to interact with p53 [29][30][31][32], we examined whether hSSB1 also associates with p53. Following co-immunoprecipitation (co-IP) using Flag-agarose or Myc-agarose, a complex containing hSSB1 and p53 was clearly detected in HEK293T cells overexpressing both exogenous hSSB1 and p53 ( Figure 2A).…”