Radioprobing the conformation of DNA in a p53-DNA complex.
OBJECTIVE
The frequency of DNA strand breaks produced by the decay of Auger electron-emitting radionuclides is inversely proportional to the distance of DNA nucleotides from the decay site; and thus is very sensitive to changes in the local conformation of the DNA. Analysis of the frequency of DNA breaks, or radioprobing, gives valuable information about the local DNA structure. More than 10 years ago, we demonstrated the feasibility of radioprobing using a DNA-repressor complex with a known structure. Herein, we used radioprobing to study the conformation of DNA in complex with the tumor suppressor protein 53 (p53). Several structures of p53-DNA complexes have been solved by X-ray crystallography. These structures, obtained with the p53 DNA binding domain, a truncated form, laid the groundwork for understanding p53-DNA interactions and their relation to p53 functions. However, whether all observed stereochemical details are relevant to the native p53-DNA complex remains unclear. A common theme of the crystallographic structures is the lack of significant bending in the central part of the DNA response element. In contrast, gel electrophoresis and electron microscopy data showed strong DNA bending and overtwisting upon binding to the native p53 tetramer.


METHODS
To analyze DNA in complex with p53, we incorporated (125)I-dCTP in two different positions of synthetic duplexes containing the consensus p53-binding site.


RESULTS
The most significant changes in the break frequency distributions were detected close to the center of the binding site, which is consistent with an increase in DNA twisting in this region and local DNA bending and sliding.


CONCLUSIONS
Our data confirm the main results of the studies made in solution and lay a foundation for systematic examination of interactions between DNA and native p53 using (125)I radioprobing.
Radiology and Imaging Sciences, Clinical Center, National Institutes of Health, Bethesda, MD, USA.
Valeri N
VN
Karamychev
Difei
D
Wang
Sharlyn J
SJ
Mazur
Ettore
E
Appella
Ronald D
RD
Neumann
Victor B
VB
Zhurkin
Igor G
IG
Panyutin
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes / Q000378:metabolism; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism*
D007457:Iodine Radioisotopes; D016159:Tumor Suppressor Protein p53; D004247:DNA