The importance of DNA structure in the regulation of basic cellular processes is an emerging field of research. Among local non-B DNA structures, inverted repeat (IR) sequences that form cruciforms and G-rich sequences that form G-quadruplexes (G4) are found in all prokaryotic and eukaryotic organisms and are targets for regulatory proteins. We analyzed IRs and G4 sequences in the genome of the most important biotechnology microorganism, S. cerevisiae. IR and G4-prone sequences are enriched in specific genomic locations and differ markedly between mitochondrial and nuclear DNA. While G4s are overrepresented in telomeres and regions surrounding tRNAs, IRs are most enriched in centromeres, rDNA, replication origins and surrounding tRNAs. Mitochondrial DNA is enriched in both IR and G4-prone sequences relative to the nuclear genome. This extensive analysis of local DNA structures adds to the emerging picture of their importance in genome maintenance, DNA replication and transcription of subsets of genes.
Non-canonical nucleic acid structures play important roles in the regulation of molecular processes. Considering the importance of the ongoing coronavirus crisis, we decided to evaluate genomes of all coronaviruses sequenced to date (stated more broadly, the order Nidovirales) to determine if they contain non-canonical nucleic acid structures. We discovered much evidence of putative G-quadruplex sites and even much more of inverted repeats (IRs) loci, which in fact are ubiquitous along the whole genomic sequence and indicate a possible mechanism for genomic RNA packaging. The most notable enrichment of IRs was found inside 5'UTR for IRs of size 12+ nucleotides, and the most notable enrichment of putative quadruplex sites (PQSs) was located before 3'UTR, inside 5'UTR, and before mRNA. This indicates crucial regulatory roles for both IRs and PQSs. Moreover, we found multiple G-quadruplex binding motifs in human proteins having potential for binding of SARS-CoV-2 RNA. Non-canonical nucleic acids structures in Nidovirales and in novel SARS-CoV-2 are therefore promising druggable structures that can be targeted and utilized in the future.
- Publikační typ
- časopisecké články MeSH
p73 is a member of the p53 protein family and has essential functions in several signaling pathways involved in development, differentiation, DNA damage responses and cancer. As a transcription factor, p73 achieves these functions by binding to consensus DNA sequences and p73 shares at least partial target DNA binding sequence specificity with p53. Transcriptional activation by p73 has been demonstrated for more than fifty p53 targets in yeast and/or human cancer cell lines. It has also been shown previously that p53 binding to DNA is strongly dependent on DNA topology and the presence of inverted repeats that can form DNA cruciforms, but whether p73 transcriptional activity has similar dependence has not been investigated. Therefore, we evaluated p73 binding to a set of p53-response elements with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures. We show by a yeast-based assay that transactivation in vivo correlated more with the relative propensity of a response element to form cruciforms than to its expected in vitro DNA binding affinity. Structural features of p73 target sites are therefore likely to be an important determinant of its transactivation function.
- MeSH
- aktivace transkripce MeSH
- konformace nukleové kyseliny MeSH
- kvasinky genetika metabolismus MeSH
- lidé MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- obrácené repetice * MeSH
- protein p73 chemie genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- vazba proteinů MeSH
- vazebná místa * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
Interferon-inducible protein 16 (IFI16) is a member of the HIN-200 protein family, containing two HIN domains and one PYRIN domain. IFI16 acts as a sensor of viral and bacterial DNA and is important for innate immune responses. IFI16 binds DNA and binding has been described to be DNA length-dependent, but a preference for supercoiled DNA has also been demonstrated. Here we report a specific preference of IFI16 for binding to quadruplex DNA compared to other DNA structures. IFI16 binds to quadruplex DNA with significantly higher affinity than to the same sequence in double stranded DNA. By circular dichroism (CD) spectroscopy we also demonstrated the ability of IFI16 to stabilize quadruplex structures with quadruplex-forming oligonucleotides derived from human telomere (HTEL) sequences and the MYC promotor. A novel H/D exchange mass spectrometry approach was developed to assess protein interactions with quadruplex DNA. Quadruplex DNA changed the IFI16 deuteration profile in parts of the PYRIN domain (aa 0-80) and in structurally identical parts of both HIN domains (aa 271-302 and aa 586-617) compared to single stranded or double stranded DNAs, supporting the preferential affinity of IFI16 for structured DNA. Our results reveal the importance of quadruplex DNA structure in IFI16 binding and improve our understanding of how IFI16 senses DNA. IFI16 selectivity for quadruplex structure provides a mechanistic framework for IFI16 in immunity and cellular processes including DNA damage responses and cell proliferation.
- MeSH
- DNA chemie genetika metabolismus MeSH
- fosfoproteiny chemie genetika metabolismus MeSH
- G-kvadruplexy * MeSH
- jaderné proteiny chemie genetika metabolismus MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- lidé MeSH
- responzivní elementy genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The breast and ovarian cancer susceptibility gene BRCA1 encodes a multifunctional tumor suppressor protein BRCA1, which is involved in regulating cellular processes such as cell cycle, transcription, DNA repair, DNA damage response and chromatin remodeling. BRCA1 protein, located primarily in cell nuclei, interacts with multiple proteins and various DNA targets. It has been demonstrated that BRCA1 protein binds to damaged DNA and plays a role in the transcriptional regulation of downstream target genes. As a key protein in the repair of DNA double-strand breaks, the BRCA1-DNA binding properties, however, have not been reported in detail. RESULTS: In this study, we provided detailed analyses of BRCA1 protein (DNA-binding domain, amino acid residues 444-1057) binding to topologically constrained non-B DNA structures (e.g. cruciform, triplex and quadruplex). Using electrophoretic retardation assay, atomic force microscopy and DNA binding competition assay, we showed the greatest preference of the BRCA1 DNA-binding domain to cruciform structure, followed by DNA quadruplex, with the weakest affinity to double stranded B-DNA and single stranded DNA. While preference of the BRCA1 protein to cruciform structures has been reported previously, our observations demonstrated for the first time a preferential binding of the BRCA1 protein also to triplex and quadruplex DNAs, including its visualization by atomic force microscopy. CONCLUSIONS: Our discovery highlights a direct BRCA1 protein interaction with DNA. When compared to double stranded DNA, such a strong preference of the BRCA1 protein to cruciform and quadruplex structures suggests its importance in biology and may thus shed insight into the role of these interactions in cell regulation and maintenance.
- MeSH
- B-DNA chemie metabolismus MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- protein BRCA1 chemie metabolismus MeSH
- proteinové domény MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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
- fluorouracil 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
The 14-3-3 protein family is a highly conserved and widely distributed group of proteins consisting of multiple isoforms in eukaryotes. Ubiquitously expressed, 14-3-3 proteins play key roles in DNA replication, cell cycle regulation, and apoptosis. The function of 14-3-3 proteins is mediated by interaction with a large number of other proteins and with DNA. It has been demonstrated that 14-3-3γ protein binds strongly to cruciform structures and is crucial for initiating replication. In this study, we analyzed DNA binding properties of the 14-3-3γ isoform to linear and supercoiled DNA. We demonstrate that 14-3-3γ protein binds strongly to long DNA targets, as evidenced by electrophoretic mobility shift assay on agarose gels. Binding of 14-3-3γ to DNA target results in the appearance of blurry, retarded DNA bands. Competition experiments with linear and supercoiled DNA on magnetic beads show very strong preference for supercoiled DNA. We also show by confocal microscopy that 14-3-3 protein in the HCT-116 cell line is co-localized with DNA cruciforms. This implies a role for the 14-3-3γ protein in its binding to local DNA structures which are stabilized by DNA supercoiling.
- MeSH
- Escherichia coli genetika MeSH
- HCT116 buňky MeSH
- klonování DNA MeSH
- kompetitivní vazba MeSH
- křížová struktura DNA genetika metabolismus MeSH
- lidé MeSH
- plazmidy genetika MeSH
- proteiny 14-3-3 genetika metabolismus MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- replikace DNA genetika MeSH
- retardační test MeSH
- superhelikální DNA genetika metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
