Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
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 (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).
Karamychev, V.N., Zhurkin, V.B., Garges, S., Neumann, R.D. & Panyutin, I.G. (1999). Detecting the DNA kinks in a DNA-CRP complex in solution with iodine-125 radioprobing. Nature Struct. Biol. 6, 747-750.
Karamychev, V.N., Wang, D., Mazur, S.J., Appella, E., Neumann, R.D., Zhurkin, V.B. & Panyutin, I.G. (2012). Radioprobing the conformation of DNA in a p53-DNA complex. Int. J. Radiat. Biol. 88, 1039-45.
Kitayner, M., Rozenberg, H., Rohs, R., Suad, O., Rabinovich, D., Honig, B. & Shakked, Z. (2010). Diversity in DNA recognition by p53 revealed by crystal structures with Hoogsteen base pairs. Nature Struct. Molec. Biol. 17, 423-429.
Sahu, G., Wang, D., Chen, C.B., Zhurkin, V.B., Harrington, R.E., Appella, E., Hager, G.L. & Nagaich, A.K. (2010). p53 binding to nucleosomal DNA depends on the rotational positioning of DNA response element. J. Biol. Chem. 285, 1321-1332.
Nagaich, A.K., Zhurkin, V.B., Durell, S.R., Jernigan, R.L., Appella, E. & Harrington, R.E. (1999). p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting. Proc. Natl. Acad. Sci. USA 96, 1875-1880.
Igor G. Panyutin1
1Radiology and Imaging Sciences