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 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.
Brázdová
Marie
M
Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolská 135, 612 65 Brno, Czech Republic.
Palecek
Jan
J
Cherny
Dmitry I
DI
Billová
Sabina
S
Fojta
Miroslav
M
Pecinka
Petr
P
Vojtesek
Borivoj
B
Jovin
Thomas M
TM
Palecek
Emil
E
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
D004278:DNA, Superhelical; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53