Journal: Nucleic Acids Research

Article Title: A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

doi: 10.1093/nar/gks1291

Figure Lengend Snippet: p53TAD2 and FPA bind to DBD-F MBP and RPA and affect DNA binding. For (A–D): Lane with DNA probe in the absence of RPA is denoted by ‘P’. ssDNA–RPA binding without FPA is marked by a (-). Concentration of labelled DNA probe is 10 nM. For (A) and (B), the concentration of FPA is 300, 100, 30, 10, 3, 1 µM, respectively. ( A ) Lack of inhibition of ssDNA–RPAΔF formation in the presence of FPA as determined by EMSA. The concentration of RPAΔF is 20 nM. ( B ) Inhibition of ssDNA–DBD-F MBP binding by FPA. The concentration of the DBD-F MBP is 2 µM. For (C) and (D), the p53TAD2 concentration is 24, 12, 6, 3, 1.5 µM, respectively. ( C ) Inhibition of helix destabilization by p53TAD2. The amount of helix destabilization was determined by the appearance of an upward shifted band consisting of RPA bound to unwound ssDNA derived from dsDNA. Lane with labelled DNA and p53TAD2 in the absence of RPA is denoted by a ‘*’. ( D ) Lack of inhibition of ssDNA–RPA binding by p53TAD2. The p53TAD2 peptide can be detected at higher concentrations due to the fluorescence of the x-rhodamine label, and does not bind DNA, as determined in figure S3C .

Article Snippet: The N-terminal X-rhodamine labelled p53TAD2, containing amino acids 40–57 (Xr-NH 2 -MDDLMLSPDDIEQWFTED), was produced by AnaSpec.

Techniques: Binding Assay, Concentration Assay, Inhibition, Derivative Assay, Fluorescence

Journal: Nucleic Acids Research

Article Title: A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

doi: 10.1093/nar/gks1291

Figure Lengend Snippet: K d values for FPA and derivatives binding to DBD-F MBP prebound to p53TAD2 as determined by fluorescence anisotropy

Article Snippet: The N-terminal X-rhodamine labelled p53TAD2, containing amino acids 40–57 (Xr-NH 2 -MDDLMLSPDDIEQWFTED), was produced by AnaSpec.

Techniques: Binding Assay, Fluorescence

Journal: Nucleic Acids Research

Article Title: A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

doi: 10.1093/nar/gks1291

Figure Lengend Snippet: K d values of DBD-F M ^B P and various RPAs for RPA and p53TAD2 substrates as determined by either fluorescence quenching or fluorescence anisotropy

Article Snippet: The N-terminal X-rhodamine labelled p53TAD2, containing amino acids 40–57 (Xr-NH 2 -MDDLMLSPDDIEQWFTED), was produced by AnaSpec.

Techniques: Fluorescence

Journal: Nucleic Acids Research

Article Title: A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

doi: 10.1093/nar/gks1291

Figure Lengend Snippet: Docking simulations of a crystal structure of DBD-F and conformer optimized for FPA binding. For both structures, FPA is shown in stick form, whereas the p53TAD2 sequence ‘MDDLMLSPDDI’ is represented by a ribbon structure. ( A ) The original crystal structure of DBD-F docked to FPA and p53TAD2 [−5.5 and −4.4 kcal/mol (24.5 µM and 204.7 µM), respectively]. ( B ) A DBD-F model optimized for FPA binding docked FPA and p53TAD2 [−6.6 and −5.9 kcal/mol (2.9 µM and 11.3 µM), respectively].

Article Snippet: The N-terminal X-rhodamine labelled p53TAD2, containing amino acids 40–57 (Xr-NH 2 -MDDLMLSPDDIEQWFTED), was produced by AnaSpec.

Techniques: Binding Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

doi: 10.1093/nar/gks1291

Figure Lengend Snippet: ( A ) Comparison of K d values for p53 peptides and FPA binding to DBD-F and RPA. Solid and hollow circles are p53TAD2 and FPA K d values, respectively, determined in this study. Solid and hollow triangles are p53TAD2b [15 aa peptide (aa 43–57 of p53)] and FPA K d values, respectively, as reported in previous literature and therein denoted by (†). ( B ) Depiction of competition between p53TAD2 and an inhibitor for the DBD-F accompanied by a change in conformation.

Article Snippet: The N-terminal X-rhodamine labelled p53TAD2, containing amino acids 40–57 (Xr-NH 2 -MDDLMLSPDDIEQWFTED), was produced by AnaSpec.

Techniques: Comparison, Binding Assay