Cruciform structure
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Interferon (IFN)-inducible HIN-200 proteins play an important role in transcriptional regulation linked to cell cycle control, inflammation, autoimmunity and differentiation. IFI16 has been identified as a target of IFNα and γ and is a member of the HIN-200 protein family. Expression level of IFI16 is often decreased in breast cancers, implicating its role as a tumor suppressor. As a potent transcription factor, IFI16 possesses a transcriptional regulatory region, a PYD/DAPIN/PAAD region which associates with IFN response, DNA-binding domains and binding regions for tumor suppressor proteins BRCA1 and p53. It is also reported that IFI16 protein is capable of binding p53 and cMYC gene promoters. Here, we demonstrate that IFI16 protein binds strongly to negatively superhelical plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of supercoiled (sc) DNA in agarose gels. Binding of IFI16 to supercoiled DNA results in the appearance of one or more retarded DNA bands on the gels. After removal of IFI16, the original mobility of the scDNA is recovered. By contrast, IFI16 protein binds very weakly to the same DNA in linear state. Using short oligonucleotide targets, we also detect a strong preference for IFI16 binding to cruciform DNA structure compared to linear DNA topology. Hence, this novel DNA-binding property of IFI16 protein to scDNA and cruciform structures may play critical roles in its tumor suppressor function.
- MeSH
- fosfoproteiny metabolismus MeSH
- jaderné proteiny metabolismus MeSH
- konformace nukleové kyseliny MeSH
- křížová struktura DNA chemie metabolismus MeSH
- lidé MeSH
- nádory genetika metabolismus MeSH
- superhelikální DNA chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cruciforms occur when inverted repeat sequences in double-stranded DNA adopt intra-strand hairpins on opposing strands. Biophysical and molecular studies of these structures confirm their characterization as four-way junctions and have demonstrated that several factors influence their stability, including overall chromatin structure and DNA supercoiling. Here, we review our understanding of processes that influence the formation and stability of cruciforms in genomes, covering the range of sequences shown to have biological significance. It is challenging to accurately sequence repetitive DNA sequences, but recent advances in sequencing methods have deepened understanding about the amounts of inverted repeats in genomes from all forms of life. We highlight that, in the majority of genomes, inverted repeats are present in higher numbers than is expected from a random occurrence. It is, therefore, becoming clear that inverted repeats play important roles in regulating many aspects of DNA metabolism, including replication, gene expression, and recombination. Cruciforms are targets for many architectural and regulatory proteins, including topoisomerases, p53, Rif1, and others. Notably, some of these proteins can induce the formation of cruciform structures when they bind to DNA. Inverted repeat sequences also influence the evolution of genomes, and growing evidence highlights their significance in several human diseases, suggesting that the inverted repeat sequences and/or DNA cruciforms could be useful therapeutic targets in some cases.
p53 Is one of the most critical proteins involved in protecting organisms from malignancies and its gene is frequently mutated in these diseases. p53 Functions as a transcription factor and its role in the cell is mediated by sequence-specific DNA binding. Although the genome contains many p53-binding sequences, the p53 protein binds only a subset of these sequences with high affinity. One likely mechanism of how p53 binds DNA effectively underlies its ability to recognize selective local DNA structure. We analyzed the possibility of cruciform structure formation within different regions of the p21 gene promoter. p53 protein remarkably activates the transcription of p21 gene after genotoxic treatment. In silico analysis showed that p21 gene promoter contains numerous p53 target sequences, some of which have inverted repeats capable of forming cruciform structures. Using chromatin immunoprecipitation, we demonstrated that p53 protein binds preferentially to sequences that not only contain inverted repeats but also have the ability to create local cruciform structures. Gel retardation assay also revealed strong preference of the p53 protein for response element in superhelical state, with cruciform structure in the DNA sequence. Taken together, our results suggest that p53 response element's potential for cruciform structure formation could be an additional determinant in p53 DNA-binding machinery.
- MeSH
- chromatinová imunoprecipitace MeSH
- fluoruracil farmakologie MeSH
- inhibitor p21 cyklin-dependentní kinasy genetika MeSH
- křížová struktura DNA genetika MeSH
- lidé MeSH
- mutageny toxicita MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- obrácené repetice genetika MeSH
- počítačová simulace MeSH
- promotorové oblasti (genetika) * MeSH
- responzivní elementy genetika MeSH
- sekvence nukleotidů MeSH
- vazba proteinů účinky léků genetika MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA cruciforms play an important role in the regulation of natural processes involving DNA. These structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling. Cruciform structures are fundamentally important for a wide range of biological processes, including replication, regulation of gene expression, nucleosome structure and recombination. They also have been implicated in the evolution and development of diseases including cancer, Werner's syndrome and others.Cruciform structures are targets for many architectural and regulatory proteins, such as histones H1 and H5, topoisomerase IIβ, HMG proteins, HU, p53, the proto-oncogene protein DEK and others. A number of DNA-binding proteins, such as the HMGB-box family members, Rad54, BRCA1 protein, as well as PARP-1 polymerase, possess weak sequence specific DNA binding yet bind preferentially to cruciform structures. Some of these proteins are, in fact, capable of inducing the formation of cruciform structures upon DNA binding. In this article, we review the protein families that are involved in interacting with and regulating cruciform structures, including (a) the junction-resolving enzymes, (b) DNA repair proteins and transcription factors, (c) proteins involved in replication and (d) chromatin-associated proteins. The prevalence of cruciform structures and their roles in protein interactions, epigenetic regulation and the maintenance of cell homeostasis are also discussed.
- MeSH
- DNA vazebné proteiny chemie metabolismus MeSH
- DNA chemie metabolismus ultrastruktura MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- molekulární sekvence - údaje MeSH
- regulace genové exprese MeSH
- replikace DNA MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
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
Cruciform structures are preferential targets for many architectural and regulatory proteins, as well as a number of DNA binding proteins with weak sequence specificity. Some of these proteins are also capable of inducing the formation of cruciform structures upon DNA binding. In this paper we analyzed the amino acid composition of eighteen cruciform binding proteins of Homo sapiens. Comparison with general amino acid frequencies in all human proteins revealed unique differences, with notable enrichment for lysine and serine and/or depletion for alanine, glycine, glutamine, arginine, tyrosine and tryptophan residues. Based on bootstrap resampling and fuzzy cluster analysis, multiple molecular mechanisms of interaction with cruciform DNA structures could be suggested, including those involved in DNA repair, transcription and chromatin regulation. The proteins DEK, HMGB1 and TOP1 in particular formed a very distinctive group. Nonetheless, a strong interaction network connecting nearly all the cruciform binding proteins studied was demonstrated. Data reported here will be very useful for future prediction of new cruciform binding proteins or even construction of predictive tool/web-based application.
- MeSH
- aminokyseliny chemie MeSH
- chromatin MeSH
- chromozomální proteiny, nehistonové chemie MeSH
- DNA vazebné proteiny chemie MeSH
- DNA-topoisomerasy I chemie MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- onkogenní proteiny chemie MeSH
- protein HMGB1 chemie MeSH
- proteiny vázající poly-ADP-ribosu chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Despite the existence of a wealth of structural and theoretical data relating to palindromic sequences in the genome, the mechanism of cruciform formation in the presence of anthracyclines in miscellaneous biological processes is still poorly understood. Generally, DNA intercalators influence the DNA superhelicity, which plays a key role in the cruciform formation in DNA molecules. The potential of DNA intercalating ligands on the stabilization/destabilization of cruciform in DNA is discussed. Here, the indirect impact of anthracyclines to cell developing and surviving is analyzed for the first time. Primarily, the anthracycline modifies the helical properties of DNA and the overall DNA structure, and secondarily alters any cruciform-dependent processes, mainly DNA replication and transcription.
The TP53 gene is the most frequently mutated gene in human cancer and p53 protein plays a crucial role in gene expression and cancer protection. Its role is manifested by interactions with other proteins and DNA. p53 is a transcription factor that binds to DNA response elements (REs). Due to the palindromic nature of the consensus binding site, several p53-REs have the potential to form cruciform structures. However, the influence of cruciform formation on the activity of p53-REs has not been evaluated. Therefore, we prepared sets of p53-REs with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures, for in vitro and in vivo analyses. Then we evaluated the presence of cruciform structures when inserted into plasmid DNA and employed a yeast-based assay to measure transactivation potential of these p53-REs cloned at a chromosomal locus in isogenic strains. We show that transactivation in vivo correlated more with relative propensity of an RE to form cruciforms than to its predicted in vitro DNA binding affinity for wild type p53. Structural features of p53-REs could therefore be an important determinant of p53 transactivation function.
DNA cruciform structures play an important role in the regulation of natural processes including gene replication and expression, as well as nucleosome structure and recombination. They have also been implicated in the evolution and development of diseases such as cancer and neurodegenerative disorders. Cruciform structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling and protein binding. They have received broad attention because of their important roles in biology. Computational approaches to study inverted repeats have allowed detailed analysis of genomes. However, currently there are no easily accessible and user-friendly tools that can analyse inverted repeats, especially among long nucleotide sequences. We have developed a web-based server, Palindrome analyser, which is a user-friendly application for analysing inverted repeats in various DNA (or RNA) sequences including genome sequences and oligonucleotides. It allows users to search and retrieve desired gene/nucleotide sequence entries from the NCBI databases, and provides data on length, sequence, locations and energy required for cruciform formation. Palindrome analyser also features an interactive graphical data representation of the distribution of the inverted repeats, with options for sorting according to the length of inverted repeat, length of loop, and number of mismatches. Palindrome analyser can be accessed at http://bioinformatics.ibp.cz.
- MeSH
- DNA bakterií analýza genetika MeSH
- DNA virů analýza genetika MeSH
- DNA analýza genetika MeSH
- Escherichia coli genetika MeSH
- genom bakteriální genetika MeSH
- genom virový genetika MeSH
- internet * MeSH
- křížová struktura DNA analýza genetika MeSH
- obrácené repetice genetika MeSH
- oligonukleotidy analýza genetika MeSH
- reprodukovatelnost výsledků MeSH
- sekvence nukleotidů MeSH
- viry klasifikace genetika MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
The RECQ4 protein belongs to the RecQ helicase family, which plays crucial roles in genome maintenance. Mutations in the RECQ4 gene are associated with three insidious hereditary disorders: Rothmund-Thomson, Baller-Gerold, and RAPADILINO syndromes. These syndromes are characterized by growth deficiency, radial ray defects, red rashes, and higher predisposition to malignancy, especially osteosarcomas. Within the RecQ family, RECQ4 is the least characterized, and its role in DNA replication and repair remains unknown. We have identified several DNA binding sites within RECQ4. Two are located at the N-terminus and one is located within the conserved helicase domain. N-terminal domains probably cooperate with one another and promote the strong annealing activity of RECQ4. Surprisingly, the region spanning 322-400aa shows a very high affinity for branched DNA substrates, especially Holliday junctions. This study demonstrates biochemical activities of RECQ4 that could be involved in genome maintenance and suggest its possible role in processing replication and recombination intermediates.
- MeSH
- helikasy RecQ chemie metabolismus MeSH
- homologní rekombinace MeSH
- křížová struktura DNA metabolismus MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- replikace DNA MeSH
- sekvence nukleotidů MeSH
- terciární struktura proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
