Detecting p53-Induced DNA Deformations by Iodine-125 Radioprobing
The tetrameric p53-DNA complex plays the key role in tumor suppression. Apparently, the fact of the p53-DNA binding becomes "known" to other members of the multi-protein cascade involved in the tumor supression pathway. Therefore, it is important to elucidate the p53-DNA complex structure, as this could lead to understanding how formation of the tetrameric complex modifies binding sites on the p53 surface (which, in turn, could serve as the signals for other proteins).
The gel electrophoresis  and electron microscopy data  demonstrate the DNA bending and increase in its twisting upon binding to p53, although the stereochemical details remain unclear. So far, the entire p53-DNA complex remains beyond the limits of NMR and X-ray crystallography. Therefore, we used a novel technique, the iodine-125 radioprobing, tested by us recently in the case of the DNA-CRP complex . The method is based on analysis of the DNA strand breaks in solution produced by the decay of an Auger electron-emitting radioisotope, 125I, incorporated in the C5 position of cytosine. We found a noticeable decrease in the frequencies of breaks in the DNA-CRP complex compared to the naked DNA duplex. This is consistent with the increase in distances between the deoxyriboses and the radioiodine atom, caused by the CRP-induced kink in the DNA. Based on these data, we establish an empirical inverse relationship between the frequencies of DNA breaks produced by 125I, and the distances from the radioisotope to the cleavage sites.
Here, to analyze the p53-DNA complex, 125I-CTP was incorporated in different positions of the duplexes containing the 20 bp long consensus p53-binding site with two tetramers CATG. The DNA break patterns proved to be clearly different for the free DNA and the p53-DNA complex, in agreement with earlier reports on the DNA deformation caused by the p53 binding [1, 2]. In particular, the decreased frequencies of DNA breaks within the consensus CATG sequence and hence, the increased sugar-iodine distances are consistent with the major-groove bends in the p53-bound DNA . This bending in the CATG tetramer is asymmetric according to our data -- the internal CA:TG dimer (located closer to the center of the 20-mer) is distorted stronger than the external CA:TG (located closer to the end of the site). In the center of the binding site, between the CATG elements, the frequencies of DNA breaks are increased, and therefore the sugar-iodine distances are decreased upon the p53 binding. This result can be interpreted as the increase in DNA twisting (or partial B-to-C transition), also in agreement with the electrophoresis data .
In conclusion, we have determined the precise locations of the DNA bending and twisting caused by the tetrameric p53 binding, and detected the difference in these deformations between the internal and the external DNA pentamers (each of which is bound to the p53 monomer). This asymmetry is interesting in the context of selectivity of the p53-DNA recognition, and formation of the new "signal" binding sites on the p53 tetrameric surface, mentioned in the beginning.
1. Nagaich, A.K., Zhurkin, V.B., Durell, S.R., Jernigan, R.L., Appella, E. and Harrington, R.E. Proc. Natl. Acad. Sci. USA 96, 1875-1880 (1999).
V. N. Karamychev, I. G. Panyutin, S. Mazur, E. Appella,
National Institutes of Health, Bethesda, MD 20892