title: Role of tumor suppressor p53 domains in selective binding to supercoiled DNA.
abstract: 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.
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolská 135, 612 65 Brno, Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Brázdová
authors.forename: Jan
authors.initials: J
authors.lastname: Palecek
authors.forename: Dmitry I
authors.initials: DI
authors.lastname: Cherny
authors.forename: Sabina
authors.initials: S
authors.lastname: Billová
authors.forename: Miroslav
authors.initials: M
authors.lastname: Fojta
authors.forename: Petr
authors.initials: P
authors.lastname: Pecinka
authors.forename: Borivoj
authors.initials: B
authors.lastname: Vojtesek
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Jovin
authors.forename: Emil
authors.initials: E
authors.lastname: Palecek
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004278:DNA, Superhelical / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D046911:Macromolecular Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D004278:DNA, Superhelical; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53