Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
EMSA detection of p53 sequence non-specific binding on PCR produced human DNA analogues
p53 is a tumor suppressor that induces cell cycle arrest and apoptosis in response to DNA damage and cancerogenesis. Its ability to bind DNA and thus play its biological role is possible in two manners. Sequence specific binding to its consensus sequence (p53CON) and sequence non-specific binding, which occures preferably to higher DNA structures. Recently has been proven, that DNA quadruplexes occure in regulation areas of most cancer important genes. In our study we have tested human DNA cloned into plasmid vectores. The DNAs were obtained by chromatin immunoprecipitation of regions which were bound by p53 with high affinity although they don't contain p53CON. Sequence studied in this work is located on noncoding region of human chromosome 7. We suggest that structure-specific binding is responsible for higher affinity of p53 binding with these areas. It has been previously found out that some single-stranded regions appeared in these areas, suggesting presence of higher DNA structures by S1 nuclease digestion (unpublished results). Because of we were unable to detect the exact location of p53 binding with sufficient resolution by standard methods we have amplified different parts of immunoprecipitated DNAs by PCR and found using EMSA to what part of the insert p53 binds with the highest affinity. This area is represented by cca 150 nucleotides. The strongest preference p53 was found for the region which contains repeated short tracts of 3-4 Ts and a short polypu.polypy sequence. It is known that dAn:dTn blocks can cause the DNA curvature and polypu.polypy sequence is able to form an intramolecular triplex
On figure a (left) you can see how the insert was divided into fragments by different primers. On figure b (middle) you can see typical reduction in band intensity vs. negative control (NC, left lane) due to binding of the protein. On figure c (right) you can see a measured band intensity decrease by ImageJ software. The work has been done in connection with project Institute of environmental technologies, reg. no. CZ.1.05/2.1.00/03.0100 supported by Research and Development for Innovations Operational Programme financed by Structural Founds of Europe Union and from the means of state budget of the Czech Republic and Czech Science Foundation (13-36108S).
1Department of Biology and Ecology, Faculty of Nature Science, University of Ostrava, Czech Republic, Chittussiho 10, 71000 Ostrava