p53 is one of the most important tumor suppressors which responds to DNA damage by binding to DNA and regulating the transcription of genes involved in cell cycle arrest, apoptosis, or senescence. As it was shown previously, p53 binding to DNA is strongly influenced by DNA topology. DNA supercoiling is fundamentally important for a wide range of biological processes including DNA transcription, replication, recombination, control of gene expression and genome organization. In this study, we investigated the cruciform structures formation of various inverted repeats in p53-responsive sequences from p21, RGC, mdm2 and GADD45 promoters under negative superhelical stress, and analyzed the effects of these DNA topology changes on p53-DNA binding. We demonstrated using three different methods (gel retardation analyses, ELISA and magnetic immunoprecipitation assay) that the p53 protein binds preferentially to negatively supercoiled plasmid DNAs with p53-responsive sequence presented as a cruciform structure. Not only the appearance of the cruciform structures within naked supercoiled DNA, but also the potential of the binding sites for adopting the non-B structures can contribute to a more favorable p53-DNA complex. Copyright 2009 Elsevier Inc. All rights reserved.
- MeSH
- buněčné linie MeSH
- ELISA MeSH
- inhibitor p21 cyklin-dependentní kinasy genetika MeSH
- intracelulární signální peptidy a proteiny genetika MeSH
- jednovláknová DNA chemie metabolismus MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- obrácené repetice MeSH
- plazmidy chemie metabolismus MeSH
- protoonkogenní proteiny c-mdm2 genetika MeSH
- regulace genové exprese MeSH
- retardační test MeSH
- sekvence nukleotidů MeSH
- superhelikální DNA chemie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
The tumour suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. P53 is a nuclear phosphoprotein and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild-type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences, differing in their internal symmetry. Binding of p53 to DNA increased with increasing negative superhelix density (-sigma). At -sigma < or = 0.03, the relative effect of DNA supercoiling on protein-DNA binding was similar for DNA containing both symmetrical and non-symmetrical target sites. On the other hand, at higher -sigma, target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding as a result of increasing levels of negative DNA supercoiling. For -sigma = 0.07, an approx. 3-fold additional increase in binding was observed for a symmetrical target site compared with a non-symmetrical target site. The p53 target sequences possessing the inverted repeat symmetry were shown to form a cruciform structure in sufficiently negative supercoiled DNA. We show that formation of cruciforms in DNA topoisomers at -sigma > or = 0.05 correlates with the extra enhancement of p53-DNA binding.
- MeSH
- biologické modely MeSH
- DNA-topoisomerasy I metabolismus MeSH
- DNA fyziologie chemie MeSH
- financování organizované MeSH
- konformace nukleové kyseliny MeSH
- kultivované buňky MeSH
- lidé MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- repetitivní sekvence nukleových kyselin MeSH
- responzivní elementy MeSH
- Spodoptera MeSH
- superhelikální DNA fyziologie chemie MeSH
- tranzitní teplota MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
