This year we celebrate seventy years since the establishment of the Institute of Biophysics of the Czechoslovak Academy of Sciences (IBP) (founded on January 1, 1955). If we look into the biography of Professor Emil Paleček (born on October 3, 1930), one of the most world-recognized personalities associated with the Institute and one of the most cited Czech scientists, known as the founder of nucleic acids electrochemistry, we are drawn to the same year, i.e. 1955, as the year in which Emil Paleček finished his studies in biochemistry and joined the IBP, where he worked with admirable vitality, enthusiasm and dedication until his death (October 30, 2018). In the context of celebration of founding of the Institute, we would like to commemorate in this article a personality who significantly influenced the history of the Institute alongside the important discoveries and research directions that defined his extremely successful career. We prefer this form, which is a sort of a mini-review of the most important results of the laboratory obtained under EP's leadership over 63 years, presented in mutual context and natural relations. For his life's work, Professor Paleček received many prestigious awards, with the Czech Head Award in 2014 and the Neuron Foundation Award in 2017 being the most distinguished.

• Keywords
• Electrochemistry, Glycans, Modification, Nucleic acids, Proteins, Structure,
• Publication type
• Journal Article MeSH
• Review MeSH

Studies have shown that 9.4 Tesla (9.4 T) high-field magnetic resonance imaging (MRI) has obvious advantages in improving image resolution and capacity, but their safety issues need to be further validated before their clinical approval. Meanwhile, emerging experimental evidences show that moderate to high intensity Static Magnetic Fields (SMFs) have some anti-cancer effects. We examined the effects of two opposite SMF directions on lung cancer bearing mice and found when the lung cancer cell-bearing mice were treated with 9.4 T SMFs for 88 h in total, the upward 9.4 T SMF significantly inhibited A549 tumor growth (tumor growth inhibition=41%), but not the downward 9.4 T SMF. In vitro cellular analysis shows that 9.4 T upward SMF treatment for 24 h not only inhibited A549 DNA synthesis, but also significantly increased ROS and P53 levels, and arrested G2 cell cycle. Moreover, the 9.4 T SMF-treatments for 88 h had no severe impairment to the key organs or blood cell count of the mice. Our findings demonstrated the safety of 9.4 T SMF long-term exposure for their future applications in MRI, and revealed the anti-cancer potential of the upward direction 9.4 T SMF.

• Keywords
• 9.4 T static magnetic field (SMF), Cell cycle, Lung cancer, P53, ROS,
• Publication type
• Journal Article MeSH

The tumor suppressor functions of p53 and its roles in regulating the cell cycle, apoptosis, senescence, and metabolism are accomplished mainly by its interactions with DNA. p53 works as a transcription factor for a significant number of genes. Most p53 target genes contain so-called p53 response elements in their promoters, consisting of 20 bp long canonical consensus sequences. Compared to other transcription factors, which usually bind to one concrete and clearly defined DNA target, the p53 consensus sequence is not strict, but contains two repeats of a 5'RRRCWWGYYY3' sequence; therefore it varies remarkably among target genes. Moreover, p53 binds also to DNA fragments that at least partially and often completely lack this consensus sequence. p53 also binds with high affinity to a variety of non-B DNA structures including Holliday junctions, cruciform structures, quadruplex DNA, triplex DNA, DNA loops, bulged DNA, and hemicatenane DNA. In this review, we summarize information of the interactions of p53 with various DNA targets and discuss the functional consequences of the rich world of p53 DNA binding targets for its complex regulatory functions.

• Keywords
• consensus sequence, cruciform, local DNA structures, p53, protein-DNA interactions,
• MeSH
• DNA chemistry   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Protein Conformation MeSH
• Consensus Sequence MeSH
• Humans MeSH
• Models, Molecular MeSH
• Tumor Suppressor Protein p53 chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA MeSH
• Tumor Suppressor Protein p53 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
• Transcriptional Activation MeSH
• Nucleic Acid Conformation MeSH
• Yeasts genetics   metabolism   MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• Inverted Repeat Sequences * MeSH
• Tumor Protein p73 chemistry   genetics   metabolism   MeSH
• Base Sequence MeSH
• Protein Binding MeSH
• Binding Sites * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH
• Tumor Protein p73 MeSH

p53 plays critical roles in regulating cell cycle, apoptosis, senescence and metabolism and is commonly mutated in human cancer. These roles are achieved by interaction with other proteins, but particularly by interaction with DNA. As a transcription factor, p53 is well known to bind consensus target sequences in linear B-DNA. Recent findings indicate that p53 binds with higher affinity to target sequences that form cruciform DNA structure. Moreover, p53 binds very tightly to non-B DNA structures and local DNA structures are increasingly recognized to influence the activity of wild-type and mutant p53. Apart from cruciform structures, p53 binds to quadruplex DNA, triplex DNA, DNA loops, bulged DNA and hemicatenane DNA. In this review, we describe local DNA structures and summarize information about interactions of p53 with these structural DNA motifs. These recent data provide important insights into the complexity of the p53 pathway and the functional consequences of wild-type and mutant p53 activation in normal and tumor cells.

• Keywords
• local DNA structures, p53 protein, protein-DNA interactions,
• MeSH
• DNA, B-Form MeSH
• DNA chemistry   genetics   metabolism   MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 chemistry   metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA, B-Form MeSH
• DNA MeSH
• Tumor Suppressor Protein p53 MeSH
• triplex DNA MeSH Browser

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
• Transcriptional Activation genetics   MeSH
• DNA-Binding Proteins chemistry   genetics   MeSH
• DNA chemistry   genetics   MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 chemistry   genetics   MeSH
• Nucleotide Motifs genetics   MeSH
• Plasmids genetics   MeSH
• Promoter Regions, Genetic MeSH
• Regulatory Sequences, Nucleic Acid genetics   MeSH
• Sequence Deletion genetics   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA MeSH
• Tumor Suppressor Protein p53 MeSH
• TP53 protein, human MeSH Browser
• triplex DNA MeSH Browser

G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320-393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this type of interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes.

• Keywords
• DNA–protein interaction, G-quadruplex, MYC, p53 protein,
• MeSH
• Circular Dichroism MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• DNA genetics   MeSH
• G-Quadruplexes * MeSH
• HCT116 Cells MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Promoter Regions, Genetic genetics   MeSH
• Proto-Oncogene Proteins c-myc genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA MeSH
• MYC protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Proto-Oncogene Proteins c-myc MeSH
• TP53 protein, human MeSH Browser

A study of the effects of salt conditions on the association and dissociation of wild type p53 with different ~3 kbp long plasmid DNA substrates (supercoiled, relaxed circular and linear, containing or lacking a specific p53 binding site, p53CON) using immunoprecipitation at magnetic beads is presented. Salt concentrations above 200 mM strongly affected association of the p53 protein to any plasmid DNA substrate. Strikingly different behavior was observed when dissociation of pre-formed p53-DNA complexes in increased salt concentrations was studied. While contribution from the p53CON to the stability of the p53-DNA complexes was detected between 100 and 170 mM KCl, p53 complexes with circular DNAs (but not linear) exhibited considerable resistance towards salt treatment for KCl concentrations as high as 2 M provided that the p53 basic C-terminal DNA binding site (CTDBS) was available for DNA binding. On the contrary, when the CTDBS was blocked by antibody used for immunoprecipitation, all p53-DNA complexes were completely dissociated from the p53 protein in KCl concentrations≥200 mM under the same conditions. These observations suggest: (a) different ways for association and dissociation of the p53-DNA complexes in the presence of the CTDBS; and (b) a critical role for a sliding mechanism, mediated by the C-terminal domain, in the dissociation process.

• MeSH
• Potassium Chloride pharmacology   MeSH
• Nucleic Acid Conformation MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• Plasmids chemistry   metabolism   MeSH
• Salts pharmacology   MeSH
• Protein Binding drug effects   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Potassium Chloride MeSH
• Tumor Suppressor Protein p53 MeSH
• Salts MeSH

Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc.), the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGG)n, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2) and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

• MeSH
• DNA-Binding Proteins chemistry   metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• G-Quadruplexes * MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• Promoter Regions, Genetic MeSH
• RNA chemistry   metabolism   MeSH
• Aging MeSH
• Telomere MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• DNA MeSH
• RNA MeSH

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
• Intracellular Signaling Peptides and Proteins 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
• Names of Substances
• Intracellular Signaling Peptides and Proteins MeSH
• Mutant Proteins MeSH
• Tumor Suppressor Protein p53 MeSH
• bcl-2-Associated X Protein MeSH
• Protein Serine-Threonine Kinases MeSH
• STK4 protein, human MeSH Browser
• DNA, Superhelical MeSH