Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Structural and Binding Properties of DNA Response Elements Bound to p53 Proteins and the Role of Spacer Sequences in p53-DNA Interactions
The tumor suppressor protein p53 binds sequence-specifically to defined DNA targets in the genome. The consensus DNA response element (RE) consists of two decameric half-sites (HS) with the general form RRRCWWGYYY (R=A,G;W=A,T;Y=C,T), separated by a variable number of base pairs (bp) (el-Deiry, 1992)). The binding of p53 to its RE is highly cooperative and relies on the recognition of DNA sequences by the core domain and on the tetramerization of the protein, which is largely mediated by DNA (Weinberg, et al., 2004; Kitayner et al., 2006; Beno et al., 2011)). Overall about 50% of all validated functional p53 REs have spacers between the half sites. An open question is whether p53 binds cooperatively to REs having spacers, especially long ones and how, or whether a different mechanism of activation exists for p53-REs with long spacers. Our cyclization kinetics experiments reveal that p53 does not induce a bend in the REs having spacers. This rules out the possibility that p53 binding results in DNA looping. We postulate that p53 incorporating both the core and tetramerization domains, referred to as p53CT (aa 94-360 of human p53), bind to REs having long spacers (10 bp or more), either hemi-specifically, with one dimer bound to a specific half-site and the other to a spacer sequence, or at a higher oligomeric state, using cooperative interactions amongst its subunits. We show that the hemi-specifically bound tetramer has higher binding affinity than the fully specific complex, however it is kinetically unstable on the DNA. Our EMSA experiments also show that the p53 binding affinity is rotationally modulated by the spacer length, i.e. it is highest for REs with 10-bp and 20-bp as spacer sequences. In addition, we show that binding of p53CT to REs, containing spacer sequences, is affected by the flexibility of the spacer sequences. These results underline the importance of cooperative interactions between p53 dimers in their binding to DNA, suggesting that the DNA response elements act as allosteric effectors in p53/DNA interactions.
Weinberg, R. L., Veprintsev, D. B., &Fersht, A. R. (2004). Cooperative binding of tetrameric p53 to DNA. Journal of molecular biology, 341(5), 1145-1159.
Kitayner, M., Rozenberg, H., Kessler, N., Rabinovich, D., Shaulov, L., Haran, T. E., & Shakked, Z. (2006). Structural basis of DNA recognition by p53 tetramers. Molecular cell, 22(6), 741-753.
Beno, I., Rosenthal, K., Levitine, M., Shaulov, L., & Haran, T. E. (2011). Sequence-dependent cooperative binding of p53 to DNA targets and its relationship to the structural properties of the DNA targets. Nucleic Acids Research, 39(5), 1919-1932.
1Department of Biology