Affinity Isolation of Activated Endogenous p53 from UV Irradiated Cultured Cells
We present a method for rapid and simple affinity isolation of endogenous, UV induced cellular p53 which possesses specific DNA binding activity. The procedure is based upon the incubation of nuclear extracts with specific sequence oligonucleotides, immobilized on magnetic beads through biotin-streptavidin interaction. Complexes are subsequently harvested, washed with the binding buffer and bound protein is eluted by elevated NaCl concentration.
Using the above described method, we have directly followed the ratio of active/inactive p53 following different UV irradiation doses and times post-irradiation. In mouse NIH3T3 fibroblasts, p53 accumulated at early ( 6 h ) timepoints after both UVC (35 J.m-2) and UVB (3500 J.m-2 ) insult showed nearly 100 % ability to bind specific DNA sequence derived from mouse p21 promoter. In contrast, at later (24 h) timepoints, p53 binding ability was dependent on the wavelength of UV used for induction: UVB induced p53 was nearly inactive, whereas UVC induction still resulted in an active p53 pool.
In human WS1 cells, these studies have been extended by using four different specific sequences: three out of them have been derived from promoters of human p53 responsive genes (p21, gadd45 and bax) and one was an artificial sequence perfectly fitting the p53 consensus. Of p53 molecules accumulated 6h after high dose of both UVB and UVC treatment, circa 50 % possessed specific binding activity towards consensus, p21 and gadd45. Interestingly, p53 that had been accumulated at late timepoint after the same treatment has lost much of its activity towards gadd45, but not towards p21 and consensus. Gadd45 responsive element sequence differs from either p21 or the synthetic consensus sequence only in a T to C transition on the eighth nucleotide position in the second halfsite. As this base is in a direct hydrogen bond contact with Cys 277 of the core domain, we speculate that, following UV treatment, this cysteine could adopt different redox states and thus altered binding ability to cytosine but not thymine. This will be tested both by using modified gadd45 responsive sequence oligo and agents modifying the p53 redox state.
Jiri Buzek and Marikki Laiho
Department of Virology, Haartman Institute, University of Helsinki,