DNA binding and exonuclease activity in the mourine alternatively-spliced p53 protein: an alternative structure and function?
The alternatively-spliced variant of the tumor-suppressor protein p53 found in mouse (referred to as AS-p53) has been the subject of biochemical and structural studies in our laboratory. In AS-p53 the last 26 amino acids of the regularly-spliced protein (referred to as RS-p53) are replaced by 17 different residues (1,2). The level of AS-p53 is comparable to that of RS-p53 and depends on the tissue and the phase of the cell cycle (3). Contrary to RS-p53, AS-p53 readily binds DNA in a sequence-specific manner without activation (4).
We have expressed AS-p53 in E. coli and purified it to homogeneity and monodispersity. The protein exhibits a high level of 3'-5' exonuclease activity as also found for RS-p53 (5). To inhibit exonuclease activity without disrupting DNA-binding activity, modified oligonucleotides where the 3'-phosphate is replaced by a phosphorothioate group were used. A quantitative gel shift assay of the AS-p53/DNA complex was developed and the dependence of the binding affinity on the presence of divalent cations and temperature was explored. Mg(II) was found essential for binding at nanomolar concentrations. An increase in the protein concentration to the micromolar range results in a nearly cooperative transition from a high-molecular-weight complex to a low-molecular-weight complex, a phenomenon that has not been observed for RS-p53. These findings suggest that AS-p53 may have a specific structural and functional role in the cell distinct from that of RS-p53.
1. Arai, N., Nomura, D., Yokota, K., Wolf, D., Brill, E., Shohat, O. and Rotter, V. Immunological distinct p53 molecules generated by alternative splicing. Mol. Cell. Biol. 6, 3232-3239 (1986).
Z. Shakked1, M. Yavnilovitch1, A.J. Gilboa1, V. Rotter2, T.E. Haran3
1Department of Structural Biology