Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed.
- MeSH
- Humans MeSH
- Mutation * MeSH
- Mutant Proteins chemistry genetics metabolism MeSH
- Cell Line, Tumor MeSH
- Tumor Suppressor Protein p53 chemistry genetics metabolism MeSH
- Plasmids genetics MeSH
- Promoter Regions, Genetic genetics MeSH
- bcl-2-Associated X Protein genetics MeSH
- Protein Serine-Threonine Kinases genetics MeSH
- Gene Expression Regulation genetics MeSH
- Substrate Specificity MeSH
- DNA, Superhelical chemistry metabolism MeSH
- Protein Binding MeSH
- Binding Sites MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
MOTIVATION: Current methods for identification of potential triplex-forming sequences in genomes and similar sequence sets rely primarily on detecting homopurine and homopyrimidine tracts. Procedures capable of detecting sequences supporting imperfect, but structurally feasible intramolecular triplex structures are needed for better sequence analysis. RESULTS: We modified an algorithm for detection of approximate palindromes, so as to account for the special nature of triplex DNA structures. From available literature, we conclude that approximate triplexes tolerate two classes of errors. One, analogical to mismatches in duplex DNA, involves nucleotides in triplets that do not readily form Hoogsteen bonds. The other class involves geometrically incompatible neighboring triplets hindering proper alignment of strands for optimal hydrogen bonding and stacking. We tested the statistical properties of the algorithm, as well as its correctness when confronted with known triplex sequences. The proposed algorithm satisfactorily detects sequences with intramolecular triplex-forming potential. Its complexity is directly comparable to palindrome searching. AVAILABILITY: Our implementation of the algorithm is available at http://www.fi.muni.cz/lexa/triplex as source code and a web-based search tool. The source code compiles into a library providing searching capability to other programs, as well as into a stand-alone command-line application based on this library. CONTACT: lexa@fi.muni.cz SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
- MeSH
- Algorithms MeSH
- Base Pair Mismatch MeSH
- DNA chemistry metabolism MeSH
- Escherichia coli K12 genetics MeSH
- Genome MeSH
- Nucleic Acid Conformation MeSH
- Humans MeSH
- Inverted Repeat Sequences MeSH
- Likelihood Functions MeSH
- Base Sequence MeSH
- Sequence Analysis, DNA methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Algorithms MeSH
- Genome MeSH
- Sequence Analysis, DNA methods MeSH
- Sequence Alignment mortality MeSH
- Software MeSH
- Publication type
- Comparative Study MeSH
