The human single-stranded DNA-binding protein, replication protein A (RPA) binds DNA in at least two different modes: initial [8±10 nucleotides (nt)] and stable (~30 nt). Switching from 8 to 30 nt mode is associated with a large conformational change. Here we report the 2.8 A Ê structure of the RPA trimerization core comprising the C-terminal DNA-binding domain of subunit RPA70 (DBD-C), the central DNA-binding domain of subunit RPA32 (DBD-D) and the entire RPA14 subunit. All three domains are built around a central oligonucleotide/oligosaccharide binding (OB)-fold and¯anked by a helix at the C-terminus. Trimerization is mediated by three C-terminal helices arranged in parallel. The OB-fold of DBD-C possesses unique structural features; embedded zinc ribbon and helix±turn±helix motifs. Using time-resolved proteolysis with trypsin, we demonstrate that the trimerization core does not contribute to the binding with substrates of 10 nt, but interacts with oligonucleotides of 24 nt. Taken together, our data indicate that switching from 8±10 to 30 nt mode is mediated by DNA binding with the trimerization core.

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...

Replication protein A (RPA), the nuclear ssDNA-binding protein in eukaryotes, is essential to DNA replication, recombination, and repair. We have shown that a globular domain at the C terminus of subunit RPA32 contains a specific surface that interacts in a similar manner with the DNA repair enzyme UNG2 and repair factors XPA and RAD52, each of which functions in a different repair pathway. NMR structures of the RPA32 domain, free and in complex with the minimal interaction domain of UNG2, were determined, defining a common structural basis for linking RPA to the nucleotide excision, base excision, and recombinational pathways of repairing damaged DNA. Our findings support a hand-off model for the assembly and coordination of different components of the DNA repair machinery.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds to and activates DNA replication from oriP, the latent origin of DNA replication in Epstein-Barr virus. The crystal structure of the DNA-binding domain of EBNA1 bound to an 18 bp binding site was solved at 2.4 A resolution. EBNA1 comprises two domains, a flanking and a core domain. The flanking domain, which includes a helix that projects into the major groove and an extended chain that travels along the minor groove, makes all of the sequence-determining contacts with the DNA. The core domain, which is structurally homologous to the complete DNA-binding domain of the bovine papilloma virus E2 protein, makes no direct contacts with the DNA bases. A model for origin unwinding is proposed that incorporates the known biochemical and structural features of the EBNA1-origin interaction.

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