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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 RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-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 oligonucleotide/oligosaccharide-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.

SUBMITTER: Bochkareva E

PROVIDER: S-EPMC1266094 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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

Bochkareva Elena E Kaustov Lilia L Ayed Ayeda A Yi Gwan-Su GS Lu Ying Y Pineda-Lucena Antonio A Liao Jack C C JC Okorokov Andrei L AL Milner Jo J Arrowsmith Cheryl H CH Bochkarev Alexey A

Proceedings of the National Academy of Sciences of the United States of America 20051017 43

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 RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-binding fold, sim ...[more]

PMID: 16234232

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Project description:UnlabelledKaposi's sarcoma-associated herpesvirus (KSHV) ORF6 is homologous to the herpes simplex virus 1 (HSV-1) ICP8 and Epstein-Barr virus (EBV) BALF2 proteins. Here, we describe its single-stranded DNA (ssDNA) binding properties. Based on previous findings with ICP8 and BALF2, a 60-amino-acid C-terminal deletion mutant of Orf6 was generated, and the protein was purified to explore the function of the C terminus in ssDNA binding. We showed that full-length ORF6 binds cooperatively to M13 ssDNA, disrupting its secondary structure and extending it to a length equivalent to that of duplex M13 DNA. The width of the ORF6-ssDNA filament is 9 nm, and a 7.3-nm repeat can be distinguished along the filament axis. Fluorescence polarization analysis revealed that the wild-type and C-terminal mutant ORF6 proteins bind equally well to short ssDNA substrates, with dissociation constant (Kd) values of 2.2 × 10(-7)M and 1.5 × 10(-7)M, respectively. These values were confirmed by electrophoretic mobility shift assay (EMSA) analysis, which also suggested that binding by the full-length protein may involve both monomers and small multimers. While no significant difference in affinities of binding between full-length ORF6 and the C-terminal deletion mutant were observed with the short DNAs, binding of the C-terminal mutant protein to M13 ssDNA showed a clear lack of cooperativity as seen by electron microscopy (EM). Incubation of a duplex DNA containing a long single-stranded tail with double-helical ORF6 protein filaments revealed that the ssDNA segment can be enveloped within the protein filament without disrupting the filament structure.ImportanceThis work describes the biochemical characterization of the single-stranded DNA binding protein of KSHV, ORF6, central to viral DNA replication in infected cells. A C-terminal deletion mutant protein was generated to aid in understanding the role of the C terminus in DNA binding. Here we analyze the binding of the wild-type and mutant proteins to short oligomeric and longer genomic ssDNA substrates. Although it is capable of interacting with the short substrates, the inability of mutant ORF6 to form oligomers in solution hindered it from fully covering the long genomic substrates. We previously showed that ORF6 forms long filaments in solution, and we showed here that these can absorb ssDNA without disruption of the filament structure. This work will provide an important basis for future studies by us and/or others.

Dataset Information

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

Publications

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

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