64 Hoogsteen or not Hoogsteen? Iodine-125 radioprobing of the p53-induced DNA deformations

Abstract

Radioprobing is suitable for tracing the DNA and RNA trajectories in nucleoprotein complexes in solution. The method is based on the analysis of the single-strand breaks produced by decay of iodine-125 incorporated in the C5 position of cytosine (Karamychev et al., 1999, 2012). Here, we used radioprobing to study the conformation of DNA in complex with the DNA binding domain (DBD) of the tumor-suppressor protein p53. Two recently crystallized DNA-p53 DBD complexes have different conformations of the CATG motifs: one with the Hoogsteen A:T pairs (Kitayner 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 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 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 are negatively correlated with the iodine–sugar distances, thus, one can evaluate the changes in these distances upon DNA binding to a protein. The radioprobing distances obtained for both DNA sequences proved to be consistent with the Watson–Crick structure observed by Chen et al. (2010). We did not find any evidence of the Hoogsteen A:T base pair formation in the DNA-p53 DBD complexes in solution using our radioprobing method. The most significant changes in the break frequency distributions were detected in the central segment of the p53 binding site, YYY|RRR, which are consistent with an increase in DNA twisting in this region and local DNA bending and sliding (Nagaich et al., 1999). We interpret these p53-induced DNA deformations in the context of p53 binding to nucleosomal DNA (Sahu et al., 2010).