Network


Latest external collaboration on country level. Dive into details by clicking on the dots.

Hotspot


Dive into the research topics where Petr Pečinka is active.

Publication


Featured researches published by Petr Pečinka.


Oncogene | 2004

Enhancement of p53 sequence-specific binding by DNA supercoiling

Emil Paleček; Václav Brázda; Eva B. Jagelská; Petr Pečinka; Lenka Karlovská; Marie Brázdová

Using a new competition assay, we investigated the effect of DNA negative supercoiling on the DNA sequence-specific binding (SSDB) of human wild-type (wt) p53 protein. We found that supercoiled (sc) pBluescript DNAs with different inserted p53 target sequences were stronger competitors than a mixture of scDNA pBluescript with the given 20-mer target oligodeoxynucleotide. ScDNAs were always better competitors than their linearized or relaxed forms. Two DNAs with extruded cruciforms within the target sequence were the best competitors; removal of the cruciforms resulted in a decrease of competitor strength. In contrast to the full-length wt p53, the deletion mutant p53CΔ30 and the p53 core domain (93–312u2009aa) showed no enhancement of p53 SSDB to scDNA, suggesting that, in addition to the p53 core domain, the C-terminal was involved in this binding. We conclude that cruciforms and DNA bends contribute to the enhancement of p53 SSDB to scDNA and that the DNA supercoiling is an important determinant in the p53 sequence-specific binding. Supercoiling may thus play a significant role in the complex p53-regulatory network.


FEBS Letters | 1990

Protonated triplex DNA in E. coli cells as detected by chemical probing

Petr Karlovsky; Petr Pečinka; M. Vojtíšková; E. Makaturová; Emil Paleček

The triplex structure in vitro is well established; however, no direct evidence has been available concerning its existence in the cell. Using the direct chemical probing here we show that the triplex H structure can exist in E. coli cells at acidic intracellular pH values; this structure differs in some details from that observed in vitro.


Biochemical Journal | 2008

DNA topology influences p53 sequence-specific DNA binding through structural transitions within the target sites

Eva B. Jagelská; Václav Brázda; Petr Pečinka; Emil Paleček; Miroslav Fojta

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.


PLOS ONE | 2013

Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

Marie Brázdová; Lucie Navrátilová; Vlastimil Tichý; Kateřina Němcová; Matej Lexa; Roman Hrstka; Petr Pečinka; Matej Adámik; Borivoj Vojtesek; Emil Paleček; Wolfgang Deppert; Miroslav Fojta

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.


Biochemical and Biophysical Research Communications | 2010

Selective binding of tumor suppressor p53 protein to topologically constrained DNA: Modulation by intercalative drugs

Hana Pivoňková; Peter Šebest; Petr Pečinka; Olga Tichá; Kateřina Němcová; Marie Brázdová; Eva B. Jagelská; Václav Brázda; Miroslav Fojta

Selective binding of the wild type tumor suppressor protein p53 to negatively and positively supercoiled (sc) DNA was studied using intercalative drugs chloroquine (CQ), ethidium bromide, acridine derivatives and doxorubicin as a modulators of the level of DNA supercoiling. The p53 was found to lose gradually its preferential binding to negatively scDNA with increasing concentrations of intercalators until the DNA negative superhelix turns were relaxed. Formation of positive superhelices (due to further increasing intercalator concentrations) rendered the circular duplex DNA to be preferentially bound by the p53 again. CQ at concentrations modulating the closed circular DNA topology did not prevent the p53 from recognizing a specific target sequence within topologically unconstrained linear DNA. Experiments with DNA topoisomer distributions differing in their superhelix densities revealed the p53 to bind selectively DNA molecules possessing higher number of negative or positive superturns. Possible modes of the p53 binding to the negatively or positively supercoiled DNA and tentative biological consequences are discussed.


FEBS Journal | 2006

DNA modification with cisplatin affects sequence-specific DNA binding of p53 and p73 proteins in a target site-dependent manner.

Hana Pivoňková; Petr Pečinka; Pavla Češková; Miroslav Fojta

Proteins p53 and p73 act as transcription factors in cell cycle control, regulation of cell development and/or in apoptotic pathways. Both proteins bind to response elements (p53 DNA‐binding sites), typically consisting of two copies of a motif RRRCWWGYYY. It has been demonstrated previously that DNA modification with the antitumor drug cisplatin inhibits p53 binding to a synthetic p53 DNA‐binding site. Here we demonstrate that the effects of global DNA modification with cisplatin on binding of the p53 or p73 proteins to various p53 DNA‐binding sites differed significantly, depending on the nucleotide sequence of the given target site. The relative sensitivities of protein–DNA binding to cisplatin DNA treatment correlated with the occurrence of sequence motifs forming stable bifunctional adducts with the drug (namely, GG and AG doublets) within the target sites. Binding of both proteins to mutated p53 DNA‐binding sites from which these motifs had been eliminated was only negligibly affected by cisplatin treatment, suggesting that formation of the cisplatin adducts within the target sites was primarily responsible for inhibition of the p53 or p73 sequence‐specific DNA binding. Distinct effects of cisplatin DNA modification on the recognition of different response elements by the p53 family proteins may have impacts on regulation pathways in cisplatin‐treated cells.


Journal of Biomolecular Structure & Dynamics | 2000

Effects of oxidation agents and metal ions on binding of p53 to supercoiled DNA

R. H. Sarma; M. H. Sarma; Miroslav Fojta; Marie Brázdová; Hana Černocká; Petr Pečinka; Václav Brázda; Jan Paleček; Eva B. Jagelská; Borivoj Vojtesek; Šárka Pospíšilová; Vinod Subramaniam; Thomas M. Jovin; Emil Paleček

Summary Wild type human full length (f.1.) tumor suppressor p53 protein binds preferentially to super-coiled (sc) DNA in vitro both in the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on the agarose gel. Bands revealed by immunoblotting with antibody DO-1 corresponded to the ethidium stained retarded bands. The intensity and the number of bands of p53-scDNA complex were decreased by physiological concentrations of unchelated zinc ions. Nickel and cobalt ions inhibited binding of p53 to scDNA and to p53CON in linear DNA fragments less efficiently than zinc. Compared to the intrinsic zinc strongly bound to Cys 176, Cys 238, Cys 242 and His 179 in the p53 core domain, binding of additional Zn2+ to p53 was much weaker as shown by an easy removal of the latter ions by low concentrations of EDTA. Oxidation of the protein with diamide resulted in a decrease of the number of the retarded bands. Under the same conditions, no binding of oxidized p53 to p53CON in a linear DNA fragment was observed. In agreement with the literature oxidation of f.1. p53 with diamide was irreversible and was not reverted by an excess of DTT. We showed that in the presence of 0.1 mM zinc ions, oxidation of p53 became reversible. Other divalent cations tested (cadmium, cobalt, nickel) exhibited no such effect. We suggested that the irreversibility of p53 oxidation was due, at least in part, to the removal of intrinsic zinc from its position in the DNA binding domain (after oxidation of the three cysteines to which the zinc ion is coordinated in the reduced protein) accompanied by a change in the p53 conformation. Binding of C-terminal anti-p53 antibody also protected bacterially expressed protein against irreversible loss of activity due to diamide oxidation. Binding the human p53 core domain (segment 94–312) to scDNA greatly differed from that observed with the full-length p53. The core domain did not posses the ability to bind strongly to many sites in scDNA regardless of the presence or absence of p53CON suggesting involvement of some other domain (probably C-terminal) in binding of the full-length p53 to scDNA. Supershift experiments using antibodies against p53 N- or C-terminus suggested that in oxidized p53, scDNA binding through the C-terminus gained importance.


Biochemical and Biophysical Research Communications | 2015

Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells.

Matej Adámik; Pavla Bažantová; Lucie Navrátilová; Alena Polášková; Petr Pečinka; Lucie Holaňová; Vlastimil Tichý; Marie Brázdová

Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA-protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA-p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed.


Journal of Biomolecular Structure & Dynamics | 1995

INTRAMOLECULAR TAT TRIPLEX IN (DA)58.(DT)58.INFLUENCE OF IONS

Petr Pečinka; D. Huertas; Fernando Azorín; Emil Paleček

Supercoil-stabilized intramolecular triplexes have been described under various conditions in different polypurine.polypyrimidine sequences such as (dG)n.(dC)n and mixed sequences including d(GA)n.d(CT)n while information about the triplexes in (dA)n.(dT)n is scarce. Using osmium tetroxide complexes and diethyl pyrocarbonate as structural probes, we show a pyrimidine.purine.pyrimidine (TAT) triplex in (dA)58.(dT)58 sequence in a supercoiled plasmid pE19. Strong modification of approximately six central thymines and approximately six Ts at the 3-end of the (dT)58 stretch as well as the DEPC modification of the 5-half of the (dA)58 strand suggested the prevalence of the H-y3 triplex conformer. At native superhelix density, optimum conditions for the triplex formation were close to 1 mM MgCl2, pH 8.5. At room temperature and MgCl2 concentrations below 0.5 and above 5 mM, almost no triplex was formed. It is suggested that the absence of the triplex at higher MgCl2 concentrations is due to the stabilization of the duplex by Mg2+ ions which prevents the duplex opening necessary for the triplex formation. At higher temperatures, favorable for duplex opening (e.g. 55 degrees C), the TAT triplex is formed even in the presence of 10 mM MgCl2. Among Ca2+, Sr2+, Ba2+, Cd2+, Zn2+ and Ni2+, only Ca2+ and Sr2+ yielded a modification pattern similar to that obtained with Mg2+; the modification pattern produced in the presence of Sr2+ was, however, much less intense. In the presence of 1 mM MgCl2, a decrease in pH from 8.5 to 7.7 resulted in a strong decrease of the triplex content. At highly negative superhelix density, the conditions for triplex formation were less stringent, and the triplex was observed even in the absence of MgCl2.


Biochimie | 2018

Bioinformatics analyses and in vitro evidence for five and six stacked G-quadruplex forming sequences

Martin Bartas; Václav Brázda; Václav Karlický; Jiří Červeň; Petr Pečinka

Quadruplexes are noncanonical DNA structures that arise in guanine rich loci and have important biological functions. Classically, quadruplexes contain four stacked intramolecular G-tetrads. Surprisingly, although some algorithms allow searching for longer than 4G tracts for quadruplex formation, these have not yet been systematically studied. Therefore, we analyzed the human genome for sequences that are predicted to adopt stacked intramolecular G-tetrads with greater than four stacks. The data provide evidence for numerous G-quadruplexes that contain five or six stacked intramolecular G-tetrads. These sequences are predominantly found in known gene regulatory regions. Electrophoretic mobility assays and circular dichroism spectroscopy indicate that these sequences form quadruplex structures inxa0vitro under physiological conditions. The localization and inxa0vitro stability of these G-quadruplexes indicate their potentially important roles in gene regulation and their potential for therapeutic applications.

Collaboration


Dive into the Petr Pečinka's collaboration.

Top Co-Authors

Avatar

Emil Paleček

Academy of Sciences of the Czech Republic

View shared research outputs
Top Co-Authors

Avatar

Miroslav Fojta

Central European Institute of Technology

View shared research outputs
Top Co-Authors

Avatar

Marie Brázdová

Academy of Sciences of the Czech Republic

View shared research outputs
Top Co-Authors

Avatar

Václav Brázda

Academy of Sciences of the Czech Republic

View shared research outputs
Top Co-Authors

Avatar

František Jelen

Academy of Sciences of the Czech Republic

View shared research outputs
Top Co-Authors

Avatar

Eva B. Jagelská

Academy of Sciences of the Czech Republic

View shared research outputs
Top Co-Authors

Avatar

Alena Kouřilová

Academy of Sciences of the Czech Republic

View shared research outputs
Top Co-Authors

Avatar
Top Co-Authors

Avatar

Kateřina Němcová

Academy of Sciences of the Czech Republic

View shared research outputs
Top Co-Authors

Avatar

Lucie Navrátilová

Academy of Sciences of the Czech Republic

View shared research outputs
Researchain Logo
Decentralizing Knowledge