Nejvíce citovaný článek - PubMed ID 21791534
A dynamic programming algorithm for identification of triplex-forming sequences
Functional DNA molecules are useful components in nanotechnology and synthetic biology. To expand the toolkit of functional DNA parts, in this study we used artificial evolution to identify a glowing deoxyribozyme called Supernova. This deoxyribozyme transfers a phosphate from a 1,2-dioxetane substrate to its 5' hydroxyl group, which triggers a chemiluminescent reaction and a flash of blue light. An engineered version of Supernova is only catalytically active in the presence of an oligonucleotide complementary to its 3' end, demonstrating that light production can be coupled to ligand binding. We anticipate that Supernova will be useful in a wide variety of applications, including as a signaling component in allosterically regulated sensors and in logic gates of molecular computers.
- Klíčová slova
- aptazyme, catalytic DNA, chemiluminescence, deoxyribozyme, in vitro selection, luminescence, sensor,
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Triplex DNA is implicated in a wide range of biological activities, including regulation of gene expression and genomic instability leading to cancer. The tumor suppressor p53 is a central regulator of cell fate in response to different type of insults. Sequence and structure specific modes of DNA recognition are core attributes of the p53 protein. The focus of this work is the structure-specific binding of p53 to DNA containing triplex-forming sequences in vitro and in cells and the effect on p53-driven transcription. This is the first DNA binding study of full-length p53 and its deletion variants to both intermolecular and intramolecular T.A.T triplexes. We demonstrate that the interaction of p53 with intermolecular T.A.T triplex is comparable to the recognition of CTG-hairpin non-B DNA structure. Using deletion mutants we determined the C-terminal DNA binding domain of p53 to be crucial for triplex recognition. Furthermore, strong p53 recognition of intramolecular T.A.T triplexes (H-DNA), stabilized by negative superhelicity in plasmid DNA, was detected by competition and immunoprecipitation experiments, and visualized by AFM. Moreover, chromatin immunoprecipitation revealed p53 binding T.A.T forming sequence in vivo. Enhanced reporter transactivation by p53 on insertion of triplex forming sequence into plasmid with p53 consensus sequence was observed by luciferase reporter assays. In-silico scan of human regulatory regions for the simultaneous presence of both consensus sequence and T.A.T motifs identified a set of candidate p53 target genes and p53-dependent activation of several of them (ABCG5, ENOX1, INSR, MCC, NFAT5) was confirmed by RT-qPCR. Our results show that T.A.T triplex comprises a new class of p53 binding sites targeted by p53 in a DNA structure-dependent mode in vitro and in cells. The contribution of p53 DNA structure-dependent binding to the regulation of transcription is discussed.
- MeSH
- aktivace transkripce genetika MeSH
- DNA vazebné proteiny chemie genetika MeSH
- DNA chemie genetika MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- nádorový supresorový protein p53 chemie genetika MeSH
- nukleotidové motivy genetika MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) MeSH
- regulační oblasti nukleových kyselin genetika MeSH
- sekvenční delece genetika MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA vazebné proteiny MeSH
- DNA MeSH
- nádorový supresorový protein p53 MeSH
- TP53 protein, human MeSH Prohlížeč
- triplex DNA MeSH Prohlížeč
Retrotransposons with long terminal repeats (LTR) form a significant proportion of eukaryotic genomes, especially in plants. They have gag and pol genes and several regulatory regions necessary for transcription and reverse transcription. We searched for potential quadruplex-forming sequences (PQSs) and potential triplex-forming sequences (PTSs) in 18 377 full-length LTR retrotransposons collected from 21 plant species. We found that PQSs were often located in LTRs, both upstream and downstream of promoters from which the whole retrotransposon is transcribed. Upstream-located guanine PQSs were dominant in the minus DNA strand, whereas downstream-located guanine PQSs prevailed in the plus strand, indicating their role both at transcriptional and post-transcriptional levels. Our circular dichroism spectroscopy measurements confirmed that these PQSs readily adopted guanine quadruplex structures-some of them were paralell-stranded, while others were anti-parallel-stranded. The PQS often formed doublets at a mutual distance of up to 400 bp. PTSs were most abundant in 3'UTR (but were also present in 5'UTR). We discuss the potential role of quadruplexes and triplexes as the regulators of various processes participating in LTR retrotransposon life cycle and as potential recombination sites during post-insertional retrotransposon-based genome rearrangements.
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
- intracelulární signální peptidy a proteiny MeSH
- lidé MeSH
- mutace * MeSH
- mutantní proteiny chemie genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 chemie genetika metabolismus MeSH
- plazmidy genetika MeSH
- promotorové oblasti (genetika) genetika MeSH
- protein X asociovaný s bcl-2 genetika MeSH
- protein-serin-threoninkinasy genetika MeSH
- regulace genové exprese genetika MeSH
- substrátová specifita MeSH
- superhelikální DNA chemie metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- intracelulární signální peptidy a proteiny MeSH
- mutantní proteiny MeSH
- nádorový supresorový protein p53 MeSH
- protein X asociovaný s bcl-2 MeSH
- protein-serin-threoninkinasy MeSH
- STK4 protein, human MeSH Prohlížeč
- superhelikální DNA MeSH
