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 major tumour suppressor protein has presented a challenge for structural biology for two decades. The intact and complete p53 molecule has eluded previous attempts to obtain its structure, largely due to the intrinsic flexibility of the protein. Using ATP-stabilised p53, we have employed cryoelectron microscopy and single particle analysis to solve the first three-dimensional structure of the full-length p53 tetramer (resolution 13.7 Å ). The p53 molecule is a D2 tetramer, resembling a hollow skewed cube with node-like vertices of two sizes. Four larger nodes accommodate central core domains, as was demonstrated by fitting of its X-ray structure. The p53 monomers are connected via their juxtaposed N-and C-termini within smaller N/C nodes to form dimers. The dimers form tetramers through the contacts between core nodes and N/C nodes. This structure revolutionises existing concepts of p53's molecular organisation and resolves conflicting data relating to its biochemical properties. This architecture of p53 in toto suggests novel mechanisms for structural plasticity, which enables the protein to bind variably spaced DNA target sequences, essential for p53 transactivation and tumour suppressor functions.

Missense point mutations can inactivate p53 and this is the most common genetic event linked with human cancer p53 is able to recognize and bind sites of DNA damage Prives, 1994;Hollstein et al., 1996). It and, in some way, damage to cellular DNA activates a is also possible that abnormal regulation of p53 may p53 response leading to G 1 arrest or apoptosis. We contribute to the development of those tumours expressing have previously shown that 'damaged DNA' induces wild-type protein (see Milner, 1995a). However, the N-terminal cleavage of p53 to generate p40(ΔN) and molecular mechanisms operating to regulate p53 are poorly p35 (core) protein products. We now show that the understood. It is likely that post-translational modification p35 product has protease activity and is able to cleave plays a regulatory role and, indeed, p53 is subject to between residues 23 and 24 of full-length p53 to numerous phosphorylations/dephosphorylations, but the generate a novel product, p50(ΔN23). This activity was functional significance of such modifications is, as yet, inhibited by bestatin, an aminopeptidase inhibitor.unclear (Meek, 1994;Shaw et al., 1996). One tantalizing Residues 23 and 24 lie within the mdm-2 binding observation is that phosphorylation at Ser315, a substrate domain of p53 and the possibility that p50(ΔN23)for CDK phosphorylation, can bias targeting of p53 to may be resistant to feedback regulation by mdm-2 specific DNA binding sequences (Wang and Prives, 1995). is discussed. Unexpectedly, interaction with ssDNA Functional regulation may also operate at the levels of induced two further cleavage products of p53, genertertiary and quaternary structure. The importance of tertiary ated by C-terminal cleavage and designated p50(ΔC) structure in p53 function is underscored by the precise and p40(ΔC). In vivo generation of a C-terminal cleavconformational folding of the core domain that is required age product of endogenous p53 similar in size to in order to position those residues involved in sequencep50(ΔC) correlated with up-regulation of p21 expresspecific DNA contacts and transactivation of p53 target sion in ML-1 cells exposed to either adriamycin or genes reviewed in Friend, 1994; Ko cisplatin. The possible significance of the various p53and Prives, 1996). Wild-type p53 has intrinsic flexibility cleavage products in relation to the cellular response and co-operative conformational shifting between monoto DNA damage is discussed. mers of oligomeric p53 has been demonstrated in vitro Keywords: activation/autoproteolysis/DNA damage/ (Milner et al., 1991) and there is evidence that p53 mdm-2/p53 conformation is subject to cell growth conditions in vivo (Milner and Watson, 1990). Additional modifications, involving N-and C-terminal epitopes of p53, are observed