Expansions of trinucleotide repeats (TNRs) are associated with genetic disorders such as Friedreich's ataxia. The tumor suppressor p53 is a central regulator of cell fate in response to different types of insults. Sequence and structure-selective modes of DNA recognition are among the main attributes of p53 protein. The focus of this work was analysis of the p53 structure-selective recognition of TNRs associated with human neurodegenerative diseases. Here, we studied binding of full length p53 and several deletion variants to TNRs folded into DNA hairpins or loops. We demonstrate that p53 binds to all studied non-B DNA structures, with a preference for non-B DNA structures formed by pyrimidine (Py) rich strands. Using deletion mutants, we determined the C-terminal DNA binding domain of p53 to be crucial for recognition of such non-B DNA structures. We also observed that p53 in vitro prefers binding to the Py-rich strand over the purine (Pu) rich strand in non-B DNA substrates formed by sequence derived from the first intron of the frataxin gene. The binding of p53 to this region was confirmed using chromatin immunoprecipitation in human Friedreich's ataxia fibroblast and adenocarcinoma cells. Altogether these observations provide further evidence that p53 binds to TNRs' non-B DNA structures.

• Keywords
• DNA hairpin, DNA–protein, frataxin, non-B DNA, p53, trinucleotide repeat,
• MeSH
• DNA chemistry   metabolism   MeSH
• Trinucleotide Repeat Expansion * MeSH
• Gene Expression MeSH
• Friedreich Ataxia genetics   metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 chemistry   metabolism   MeSH
• Pyrimidines MeSH
• Recombinant Proteins MeSH
• Trinucleotide Repeats * MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH
• Tumor Suppressor Protein p53 MeSH
• Pyrimidines MeSH
• Recombinant Proteins MeSH
• TP53 protein, human MeSH Browser

Presence of mutated and/or structurally modified (e.g., denatured, aggregated) protein p53 form is associated with several disorders such as Alzheimer's disease, Parkinson's disease, prion diseases, and many types of tumours. The aim of this work was to distinguish native, denatured and aggregated form of full-length p53 by flow injection analysis coupled with electrochemical detector (FIA-ED). Firstly FIA-ED method used for protein native form determination was optimized (detection limit 45.8 amol per 5 mul injection; 3 x S/N). In addition the technique was applied to identify p53 structural forms (denatured and aggregated). It was found out that denatured form provides about three times higher electrochemical response (protein structure unfolding, approach of more electroactive centers - aminoacid residues - towards electrode surface) in comparison with native form. On the other hand, aggregated form offers lower response (steric eclipse of electroactive protein parts) when compared with the signal of native form. The obtained data show that we are not only able to sensitively determine native, denatured, and aggregated structural forms of p53 protein but also to distinguish them.

• MeSH
• Protein Denaturation MeSH
• Electrochemistry MeSH
• Protein Conformation MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 chemistry   MeSH
• Flow Injection Analysis methods   MeSH
• Sensitivity and Specificity MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH

We showed previously that bacterially expressed full-length human wild-type p53b(1-393) binds selectively to supercoiled (sc)DNA in sc/linear DNA competition experiments, a process we termed supercoil-selective (SCS) binding. Using p53 deletion mutants and pBluescript scDNA (lacking the p53 recognition sequence) at native superhelix density we demonstrate here that the p53 C-terminal domain (amino acids 347-382) and a p53 oligomeric state are important for SCS binding. Monomeric p53(361-393) protein (lacking the p53 tetramerization domain, amino acids 325-356) did not exhibit SCS binding while both dimeric mutant p53(319- 393)L344A and fusion protein GCN4-p53(347-393) were effective in SCS binding. Supershifting of p53(320-393)-scDNA complexes with monoclonal antibodies revealed that the amino acid region 375-378, constituting the epitope of the Bp53-10.1 antibody, plays a role in binding of the p53(320-393) protein to scDNA. Using electron microscopy we observed p53-scDNA nucleoprotein filaments produced by all the C-terminal proteins that displayed SCS binding in the gel electrophoresis experiments; no filaments formed with the monomeric p53(361- 393) protein. We propose a model according to which two DNA duplexes are compacted into p53-scDNA filaments and discuss a role for filament formation in recombination.

• MeSH
• Models, Biological MeSH
• Humans MeSH
• Macromolecular Substances MeSH
• Molecular Sequence Data MeSH
• Tumor Suppressor Protein p53 chemistry   metabolism   ultrastructure   MeSH
• Recombinant Fusion Proteins metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Deletion MeSH
• DNA, Superhelical metabolism   ultrastructure   MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Macromolecular Substances MeSH
• Tumor Suppressor Protein p53 MeSH
• Recombinant Fusion Proteins MeSH
• DNA, Superhelical MeSH