title: Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library.
abstract: Temperature-sensitive (ts) mutations have been used as a genetic and molecular tool to study the functions of many gene products. Each ts mutant protein may contain a temperature-dependent intramolecular mechanism such as ts conformational change. To identify key ts structural elements controlling the protein function, we screened ts p53 mutants from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae. We isolated 142 ts p53 mutants, including 131 unreported ts mutants. These mutants clustered in beta-strands in the DNA-binding domain, particularly in one of the two beta-sheets of the protein, and 15 residues (Thr155, Arg158, Met160, Ala161, Val172, His214, Ser215, Pro223, Thr231, Thr253, Ile254, Thr256, Ser269, Glu271, and Glu285) were ts hot spots. Among the 142 mutants, 54 were examined further in human osteosarcoma Saos-2 cells, and it was confirmed that 89% of the mutants were also ts in mammalian cells. The ts mutants represented distinct ts transactivities for the p53 binding sequences and a distinct epitope expression pattern for conformation-specific anti-p53 antibodies. These results indicated that the intramolecular beta-sheet in the core DNA-binding domain of p53 was a key structural element controlling the protein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function.
authors.affiliation: Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan.
authors.forename: Kazuko
authors.initials: K
authors.lastname: Shiraishi
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kato
authors.forename: Shuang-Yin
authors.initials: SY
authors.lastname: Han
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.forename: Kazunori
authors.initials: K
authors.lastname: Otsuka
authors.forename: Masato
authors.initials: M
authors.lastname: Sakayori
authors.forename: Takanori
authors.initials: T
authors.lastname: Ishida
authors.forename: Motohiro
authors.initials: M
authors.lastname: Takeda
authors.forename: Ryunosuke
authors.initials: R
authors.lastname: Kanamaru
authors.forename: Noriaki
authors.initials: N
authors.lastname: Ohuchi
authors.forename: Chikashi
authors.initials: C
authors.lastname: Ishioka
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D015723:Gene Library; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013050:Spectrometry, Fluorescence; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA