Hoogsteen or not Hoogsteen? Iodine-125 Radioprobing of the P53-Induced DNA Deformations

Radioprobing is suitable for tracing the DNA and RNA trajectories in nucleoprotein complexes in solution. The method is based on analysis of the single-strand breaks produced by decay of iodine-125 incorporated in the C5 position of cytosine.Two recently crystallized p53-DNA complexes have different conformations of the CATG motifs: one with the Hoogsteen A:T pairs (Shakked et al., 2010) and the other with the Watson-Crick pairs (Chen et al., 2010). The two complexes differ in the sequence of the central YYY|RRR junction: the first one has the C|G step and the second one has the T|A step.Thus, it is interesting to apply the radioprobing method to the two DNA sequences used in crystallography, to see if the local changes (T|A to C|G) in the center of the p53 response element would produce significant distortions in the CATG motifs. To this aim, the iodine-containing cytosine, C∗, was incorporated in the duplexes containing p53-binding sites, in one of the two CATG motifs, and the frequencies of DNA breaks were analyzed. Frequencies of breaks negatively correlate with the iodine-sugar distances, therefore, one can evaluate the changes in DNA conformation upon binding to a protein. The radioprobing distances thus obtained proved to be consistent with the Watson-Crick structure observed by Chen et al. That is, our data testify against formation of the Hoogsteen A:T base pairs -- the C∗ATG motifs retain canonical B-DNA conformation in both DNA sequences.The most significant changes in the break frequency distributions were detected in the central segment of the p53 binding site, YYY|RRR, which is consistent with an increase in DNA twisting in this region and local DNA bending and sliding. We interpret these p53-induced DNA deformations in the context of p53 binding to nucleosomal DNA.