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

Brazdova, M.; Palecek, J.; Cherny, D. I.; Billova, S.; Fojta, M.; Pecinka, P.; Vojtesek, B.; Jovin, T. M.; Palecek, E.

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

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Cherny, D. I.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

Jovin, T. M.
Max Planck Society;

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引用

Brazdova, M., Palecek, J., Cherny, D. I., Billova, S., Fojta, M., Pecinka, P., Vojtesek, B., Jovin, T. M., & Palecek, E. (2002). Role of tumor suppressor p53 domains in selective binding to supercoiled DNA. Nucleic Acids Research, 30(22), 4966-4974. Retrieved from http://nar.oxfordjournals.org/cgi/reprint/30/22/4966.

引用: https://hdl.handle.net/11858/00-001M-0000-0012-F291-0

要旨

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.