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 is a nuclear phosphoprotein that regulates cellular fate after genotoxic stress through its role as a transcriptional regulator of genes involved in cell cycle control and apoptosis. The C-terminal region of p53 is known to negatively regulate sequence specific DNA-binding of p53; modifications to the C-terminus relieve this inhibition. Two models have been proposed to explain this latency: (i) an allosteric model in which the C-terminal domain interacts with another domain of p53 or (ii) a competitive model in which the C-terminal and the core domains compete for DNA binding. We have characterized latent and active forms of dimeric p53 using gel mobility shift assays and NMR spectroscopy. We show on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that the C-terminus does not interact with other p53 domains. Similarly, NMR spectra of isolated core and tetramerization domains confirm a modular p53 architecture. The data presented here rule out an allosteric model for the regulation of p53.

The structure of 21-residue antimicrobial peptide buforin II has been determined by using NMR spectroscopy and restrained molecular dynamics. Buforin II adopts a flexible random structure in H20. In trifluoroethanol (TFE)/H20 (1 : 1, vlvi mixture, however, buforin II assumes a regular m-helix between residues Va112 and Arg 2° and a distorted helical structure between residues Gly 7 and Pro n. The model structure obtained shows an amphipathic character in the region from Arg 5 to the C-terminus, Lys 2z. Like other known cationic antimicrobial peptides, the amphipathic structure might be the key factor for antimicrobial activity of buforin II.

Emerging molecular diagnosis requires ultrafast polymerase chain reaction (PCR) on chip for rapid precise detection of infectious diseases in the point-of-care test. Here, we report nanoplasmonic on-chip PCR for rapid precision molecular diagnostics. The nanoplasmonic pillar arrays (NPA) comprise gold nanoislands on the top and sidewall of large-scale glass nanopillar arrays. The nanoplasmonic pillars enhance light absorption of a white light-emitting diode (LED) over the whole visible range due to strong electromagnetic hotspots between the nanoislands. As a result, they effectively induce photothermal heating for ultrafast PCR thermal cycling. The temperature profile of NPA exhibits 30 cycles between 98 and 60 °C for a total of 3 min and 30 s during the cyclic excitation of white LED light. The experimental results also demonstrate the rapid DNA amplification of both 0.1 ng μL −1 of λ-DNA in 20 thermal cycles and 0.1 ng μL −1 of complementary DNA of Middle East respiratory syndrome coronavirus in 30 thermal cycles using a conventional PCR volume of 15 μL. This nanoplasmonic PCR technique provides a new opportunity for rapid precision molecular diagnostics.