Role of tumor suppressor p53 domains in selective binding to supercoiled DNA

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 recognit...

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Bibliographic Details
Published in:Nucleic acids research 2002-11, Vol.30 (22), p.4966-4974
Main Authors: Brázdová, Marie, Paleček, Jan, Cherny, Dmitry I., Billová, Sabina, Fojta, Miroslav, Pečinka, Petr, Vojtěšek, Bořivoj, Jovin, Thomas M., Paleček, Emil
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Binding Sites
DNA, Superhelical - metabolism
DNA, Superhelical - ultrastructure
Humans
Macromolecular Substances
Models, Biological
Molecular Sequence Data
Protein Binding
Protein Structure, Tertiary
Recombinant Fusion Proteins - metabolism
Sequence Deletion
Tumor Suppressor Protein p53 - chemistry
Tumor Suppressor Protein p53 - metabolism
Tumor Suppressor Protein p53 - ultrastructure
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Summary: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.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gkf616