Differential interaction of wild type and mutant p53 to promoter sequences and analysis of interacting proteins.

摘要

The p53 tumor suppressor gene is the most common mutated gene in cancer. Cancer-associated mutations in the p53 gene often change amino acids in the protein's DNA binding domain. This research tries to analyze the DNA binding of the tumor suppressor p53 from 4 human cell lines with the mdm2, p21 and cyclin G gene regulatory sequence and a mutant form of the cyclin G sequence. Treatment of MCF-7 cells having wild type p53 with hydrogen peroxide increased the binding of p53 to DNA. The mutant p53 from thyroid cancer cell lines showed differential interaction of p53 to different gene regulatory sequences. This supports the role of p53 protein as an oncogene, where the mutant p53 protein represses wild type p53 regulated genes. To better understand how p53 protein regulates different response elements, an attempt to study the proteins interacting with DNA bound p53 has been done. Using streptavidin magnetic beads and biotinylated DNA, DNA bound p53 and associated proteins could be isolated. Proteins known to be associated with p53 could not be detected using western blots in the bound fractions however different protein spots were visible using 2D gels. Differences were observed when wild type and mutant p53 proteins were bound to wild type and mutant cyclin G sequence. This method gives a way to purify the DNA bound p53 associated proteins which may be important for p53 gene regulation. The research also shows potential colorimetric detection method for DNA binding proteins and restriction enzymes digestion of DNA assembled to gold nanoparticles. The results for this approach were not conclusive but a shift in the DNA was observed. Overall this research provides new insights into the p53 protein biology by understanding DNA binding by this protein.