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1.
Anal Chem ; 93(29): 10084-10089, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34264066

RESUMO

We report for the first time on in situ transduction of electrochemical responses of ion-selective electrodes, operating under non-zero-current conditions, to emission change signals. The proposed novel-type PVC-based membrane comprises a dispersed redox and emission active ion-to-electron transducer. The electrochemical trigger applied induces a redox process of the transducer, inducing ion exchange between the membrane and the solution, resulting also in change of its emission spectrum. It is shown that electrochemical signals recorded for ion-selective electrodes operating under voltammetric/coulometric conditions correlate with emission intensity changes recorded in the same experiments. Moreover, the proposed optical readout offers extended linear response range compared to electrical signals recorded in voltammetric or coulometric mode.


Assuntos
Elétrons , Eletrodos Íon-Seletivos , Eletrodos , Oxirredução
2.
BMC Res Notes ; 14(1): 275, 2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34281605

RESUMO

OBJECTIVE: The ability to form nucleoprotein complexes is a fundamental activity of DNA replication initiation proteins. They bind within or nearby the region of replication origin what results in melting of a double-stranded DNA (dsDNA) and formation of single-stranded DNA (ssDNA) region where the replication machinery can assemble. For prokaryotic initiators it was shown that they interact with the formed ssDNA and that this interaction is required for the replication activity. The ability to interact with ssDNA was also shown for Saccharomyces cerevisiae replication initiation protein complex ORC. For Archaea, which combine features of both prokaryotic and eukaryotic organisms, there was no evidence whether DNA replication initiators can interact with ssDNA. We address this issue in this study. RESULTS: Using purified Orc1 protein from Aeropyrum pernix (ApOrc1) we analyzed its ability to interact with ssDNA containing sequence of an AT-rich region of the A. pernix origin Ori1 as well as with homopolymers of thymidine (polyT) and adenosine (polyA). The Bio-layer interferometry, surface plasmon resonance and microscale thermophoresis showed that the ApOrc1 can interact with ssDNA and it binds preferentially to T-rich ssDNA. The hydrolysis of ATP is not required for this interaction.


Assuntos
DNA de Cadeia Simples , Complexo de Reconhecimento de Origem , Archaea/metabolismo , Replicação do DNA , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Ligação Proteica , Origem de Replicação
3.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806461

RESUMO

The present study aimed to synthesize novel polycationic polymers composed of N-substituted L-2,3-diaminopropionic acid residues (DAPEGs) and investigate their cell permeability, cytotoxicity, and DNA-binding ability. The most efficient cell membrane-penetrating compounds (O2Oc-Dap(GO2)n-O2Oc-NH2, where n = 4, 6, and 8) showed dsDNA binding with a binding constant in the micromolar range (0.3, 3.4, and 0.19 µM, respectively) and were not cytotoxic to HB2 and MDA-MB-231 cells. Selected compounds used in the transfection of a GFP plasmid showed high transfection efficacy and minimal cytotoxicity. Their interaction with plasmid DNA and the increasing length of the main chain of tested compounds strongly influenced the organization and shape of the flower-like nanostructures formed, which were unique for 5/6-FAM-O2Oc-[Dap(GO2)]8-O2Oc-NH2 and typical for large proteins.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Ácidos Nucleicos/metabolismo , Polímeros/farmacologia , beta-Alanina/análogos & derivados , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Nanoestruturas/química , Plasmídeos/metabolismo , Transfecção/métodos , beta-Alanina/farmacologia
4.
Nucleic Acids Res ; 49(6): 3394-3408, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33660784

RESUMO

An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA-protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Transativadores/química , Transativadores/metabolismo , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
5.
J Orthop Trauma ; 34 Suppl 2: S19-S20, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32639343

RESUMO

This case demonstrates a recognized association between an acetabular injury pattern and underlying morphology of the hip. In the patient discussed, hyperflexion of the hip results in the engagement of the present CAM lesion, and the resulting subluxation leads to a fracture of the posterior wall and instability of the hip. This combination of pathologies was addressed with a surgical dislocation approach to address both the CAM lesion and fix the posterior wall.


Assuntos
Fraturas Ósseas , Luxação do Quadril , Acetábulo/diagnóstico por imagem , Acetábulo/cirurgia , Luxação do Quadril/diagnóstico por imagem , Luxação do Quadril/etiologia , Luxação do Quadril/cirurgia , Humanos
6.
Int J Mol Sci ; 21(2)2020 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-31963646

RESUMO

Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14-39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.


Assuntos
Dissulfetos/química , Peptídeos/farmacologia , Receptores Imunológicos/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Receptores Imunológicos/química , Membro 14 de Receptores do Fator de Necrose Tumoral/química
7.
Sci Rep ; 8(1): 15287, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30327496

RESUMO

The activity of type II toxin-antitoxin systems (TA), which are responsible for many important features of bacterial cells, is based on the differences between toxin and antitoxin stabilities. The antitoxin lability results from bacterial protease activity. Here, we investigated how particular Escherichia coli cytosolic proteases, namely, Lon, ClpAP, ClpXP, and ClpYQ, affect the stability of both the toxin and antitoxin components of the parDE system from the broad host range plasmid RK2. The results of our in vivo and in vitro experiments show that the ParD antitoxin is degraded by the ClpAP protease, and dsDNA stimulates this process. The ParE toxin is not degraded by any of these proteases and can therefore cause growth inhibition of plasmid-free cells after an unequal plasmid distribution during cell division. We also demonstrate that the ParE toxin interaction with ParD prevents antitoxin proteolysis by ClpAP; however, this interaction does not prevent the ClpAP interaction with ParD. We show that ClpAP protease homologs affect plasmid stability in other bacterial species, indicating that ClpAP is a universal activator of the parDE system and that ParD is a universal substrate for ClpAP.


Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Sistemas Toxina-Antitoxina , Caulobacter crescentus/genética , DNA/fisiologia , Escherichia coli/enzimologia , Escherichia coli/genética , Plasmídeos , Ligação Proteica , Proteólise , Pseudomonas putida/genética
8.
Nucleic Acids Res ; 45(7): 3953-3966, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28335002

RESUMO

Specific nucleoprotein complexes are formed strictly to prevent over-initiation of DNA replication. An example of those is the so-called handcuff complex, in which two plasmid molecules are coupled together with plasmid-encoded replication initiation protein (Rep). In this work, we elucidate the mechanism of the handcuff complex disruption. In vitro tests, including dissociation progress analysis, demonstrate that the dimeric variants of plasmid RK2 replication initiation protein TrfA are involved in assembling the plasmid handcuff complex which, as we found, reveals high stability. Particular proteases, namely Lon and ClpAP, disrupt the handcuff by degrading TrfA, thus affecting plasmid stability. Moreover, our data demonstrate that TrfA monomers are able to dissociate handcuffed plasmid molecules. Those monomers displace TrfA molecules, which are involved in handcuff formation, and through interaction with the uncoupled plasmid replication origins they re-initiate DNA synthesis. We discuss the relevance of both Rep monomers and host proteases for plasmid maintenance under vegetative and stress conditions.


Assuntos
Replicação do DNA , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Plasmídeos/biossíntese , Protease La/metabolismo , DNA Bacteriano/biossíntese , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Nucleoproteínas/metabolismo , Plasmídeos/metabolismo , Protease La/genética , Multimerização Proteica
9.
J Biol Chem ; 292(18): 7507-7518, 2017 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-28292931

RESUMO

Lon protease previously has been shown to interact with DNA, but the role of this interaction for Lon proteolytic activity has not been characterized. In this study, we used truncated Escherichia coli Lon constructs, bioinformatics analysis, and site-directed mutagenesis to identify Lon domains and residues crucial for Lon binding with DNA and effects on Lon proteolytic activity. We found that deletion of Lon's ATPase domain abrogated interactions with DNA. Substitution of positively charged amino acids in this domain in full-length Lon with residues conferring a net negative charge disrupted binding of Lon to DNA. These changes also affected the degradation of nucleic acid-binding protein substrates of Lon, intracellular localization of Lon, and cell morphology. In vivo tests revealed that Lon-DNA interactions are essential for Lon activity in cell division control. In summary, we demonstrate that the ability of Lon to bind DNA is determined by its ATPase domain, that this binding is required for processing protein substrates in nucleoprotein complexes, and that Lon may help regulate DNA replication in response to growth conditions.


Assuntos
Adenosina Trifosfatases/metabolismo , Replicação do DNA/fisiologia , DNA Bacteriano/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Protease La/metabolismo , Adenosina Trifosfatases/genética , Divisão Celular/fisiologia , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Protease La/genética , Domínios Proteicos
10.
Front Mol Biosci ; 3: 39, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27563644

RESUMO

The proper initiation and occurrence of DNA synthesis depends on the formation and rearrangements of nucleoprotein complexes within the origin of DNA replication. In this review article, we present the current knowledge on the molecular mechanism of replication complex assembly at the origin of bacterial chromosome and plasmid replicon containing direct repeats (iterons) within the origin sequence. We describe recent findings on chromosomal and plasmid replication initiators, DnaA and Rep proteins, respectively, and their sequence-specific interactions with double- and single-stranded DNA. Also, we discuss the current understanding of the activities of DnaA and Rep proteins required for replisome assembly that is fundamental to the duplication and stability of genetic information in bacterial cells.

11.
Plasmid ; 86: 7-13, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27252071

RESUMO

Plasmids, as extrachromosomal genetic elements, need to work out strategies that promote independent replication and stable maintenance in host bacterial cells. Their maintenance depends on constant formation and dissociation of nucleoprotein complexes formed on plasmid DNA. Plasmid replication initiation proteins (Rep) form specific complexes on direct repeats (iterons) localized within the plasmid replication origin. Formation of these complexes along with a strict control of Rep protein cellular concentration, quaternary structure, and activity, is essential for plasmid maintenance. Another important mechanism for maintenance of low-copy-number plasmids are the toxin-antitoxin (TA) post-segregational killing (psk) systems, which prevent plasmid loss from the bacterial cell population. In this mini review we discuss the importance of nucleoprotein complex processing by energy-dependent host proteases in plasmid DNA replication and plasmid type II toxin-antitoxin psk systems, and draw attention to the elusive role of DNA in this process.


Assuntos
Antitoxinas/genética , Toxinas Bacterianas/genética , DNA Bacteriano/genética , Escherichia coli/genética , Plasmídeos/genética , Proteólise , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Origem de Replicação/genética
12.
PLoS One ; 10(2): e0117413, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25710793

RESUMO

Bacterial HtrAs are proteases engaged in extracytoplasmic activities during stressful conditions and pathogenesis. A model prokaryotic HtrA (HtrA/DegP from Escherichia coli) requires activation to cleave its substrates efficiently. In the inactive state of the enzyme, one of the regulatory loops, termed LA, forms inhibitory contacts in the area of the active center. Reduction of the disulfide bond located in the middle of LA stimulates HtrA activity in vivo suggesting that this S-S bond may play a regulatory role, although the mechanism of this stimulation is not known. Here, we show that HtrA lacking an S-S bridge cleaved a model peptide substrate more efficiently and exhibited a higher affinity for a protein substrate. An LA loop lacking the disulfide was more exposed to the solvent; hence, at least some of the interactions involving this loop must have been disturbed. The protein without S-S bonds demonstrated lower thermal stability and was more easily converted to a dodecameric active oligomeric form. Thus, the lack of the disulfide within LA affected the stability and the overall structure of the HtrA molecule. In this study, we have also demonstrated that in vitro human thioredoxin 1 is able to reduce HtrA; thus, reduction of HtrA can be performed enzymatically.


Assuntos
Escherichia coli/enzimologia , Proteínas de Choque Térmico/metabolismo , Proteínas Periplásmicas/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Dissulfetos , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Histidina/genética , Histidina/metabolismo , Cinética , Dados de Sequência Molecular , Mutagênese , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Oxirredução , Proteínas Periplásmicas/química , Proteínas Periplásmicas/genética , Proteólise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Alinhamento de Sequência , Serina Endopeptidases/química , Serina Endopeptidases/genética , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Temperatura
13.
Plasmid ; 76: 72-8, 2014 11.
Artigo em Inglês | MEDLINE | ID: mdl-25454070

RESUMO

DNA replication initiation has been well-characterized; however, studies in the past few years have shown that there are still important discoveries to be made. Recent publications concerning the bacterial DnaA protein have revealed how this replication initiator, via interaction with specific sequences within the origin region, causes local destabilization of double stranded DNA. Observations made in the context of this bacterial initiator have also been converging with those recently made for plasmid Rep proteins. In this mini review we discuss the relevance of new findings for the RK2 plasmid replication initiator, TrfA, with regard to new data on the structure of complexes formed by the chromosomal replication initiator DnaA. We discuss structure-function relationships of replication initiation proteins.


Assuntos
Proteínas de Escherichia coli/metabolismo , Plasmídeos/genética , Origem de Replicação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética
14.
Nucleic Acids Res ; 42(12): 7807-18, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24838560

RESUMO

The DNA unwinding element (DUE) is a sequence rich in adenine and thymine residues present within the origin region of both prokaryotic and eukaryotic replicons. Recently, it has been shown that this is the site where bacterial DnaA proteins, the chromosomal replication initiators, form a specific nucleoprotein filament. DnaA proteins contain a DNA binding domain (DBD) and belong to the family of origin binding proteins (OBPs). To date there has been no data on whether OBPs structurally different from DnaA can form nucleoprotein complexes within the DUE. In this work we demonstrate that plasmid Rep proteins, composed of two Winged Helix domains, distinct from the DBD, specifically bind to one of the strands of ssDNA within the DUE. We observed nucleoprotein complexes formed by these Rep proteins, involving both dsDNA containing the Rep-binding sites (iterons) and the strand-specific ssDNA of the DUE. Formation of these complexes required the presence of all repeated sequence elements located within the DUE. Any changes in these repeated sequences resulted in the disturbance in Rep-ssDNA DUE complex formation and the lack of origin replication activity in vivo or in vitro.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Plasmídeos/genética , Origem de Replicação , Sequência Rica em At , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/química , DNA de Cadeia Simples/química
15.
Microbiol Spectr ; 2(6)2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26104462

RESUMO

Iteron-containing plasmids are model systems for studying the metabolism of extrachromosomal genetic elements in bacterial cells. Here we describe the current knowledge and understanding of the structure of iteron-containing replicons, the structure of the iteron plasmid encoded replication initiation proteins, and the molecular mechanisms for iteron plasmid DNA replication initiation. We also discuss the current understanding of control mechanisms affecting the plasmid copy number and how host chaperone proteins and proteases can affect plasmid maintenance in bacterial cells.


Assuntos
Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Plasmídeos , Sequências Repetitivas de Ácido Nucleico/genética
16.
Przegl Lek ; 71(12): 666-71, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25951693

RESUMO

BACKGROUND: In most cases the only knowledge an individual will receive with regards to their own body and its proper functioning is during their high school education. The aim of this study was to evaluate high school students' knowledge about basic physiology. MATERIAL AND METHODS: The research was carried out in five, randomly chosen high schools in Krakow, Poland. Young people in the age of 17-19 years were asked to fill in the questionnaire designed by the authors. The first part of the survey included personal data. The second part contained 20 close-ended questions assessing students' knowledge about the basics of human physiology. Question difficulty varied from easy through average, and up to difficult. The maximum number of points to achieve was 20. RESULTS: One-thousand-and eighty-three (out of 1179 invited--91.86%) Polish high school students (63.25% female) filled in a 20-item questionnaire constructed by the authors regarding basic human physiology. The mean age of the group was 17.66 ± 0.80 years. The mean score among the surveyed was 10.15 ± 3.48 (range 0-20). Only 26.04% of students achieved a grade of 60% or more, and only one person obtained the highest possible score. Females achieved significantly better scores than males (10.49 ± 3.38 vs. 9.56 ± 3.56; p < 0.0001). Pupils in their second year who were in the process of studying physiology, obtained better results than those in their third year who had already finished the biology course (10.70 ± 3.27 vs. 9.81 ± 3.74 respectively; p < 0.0001) and those in their first year who did not yet study human physiology (10.70 ± 3.27 vs. 9.63 ± 2.74 respectively; p = 0.003). Over 23% of students did not know that mature red blood cells do not have cell nuclei and a similar number of them answered that humans have 500,000 erythrocytes in 1 mm3 of blood. Over 32% believed that plasma does not participate in the transport of respiratory gases, and 31% believed that endocrine glands secrete hormones within their immediate vicinity and into the blood. CONCLUSIONS: Our research has shown that young people, especially men, often lack basic physiological knowledge needed to make conscious and responsible decisions regarding their health. Our results suggest that more emphasis should be put on properly teaching human physiology in high school, especially to those students who do not plan a career in medicine-related fields. This study brings to light the disturbing fact that about a year after a student finishes his basic physiology course his knowledge of the subject returns to a pre high school level.


Assuntos
Avaliação Educacional/estatística & dados numéricos , Conhecimentos, Atitudes e Prática em Saúde , Fisiologia/educação , Estudantes/estatística & dados numéricos , Adolescente , Estudos Transversais , Escolaridade , Feminino , Humanos , Masculino , Polônia , Inquéritos e Questionários , Adulto Jovem
17.
Microbiology (Reading) ; 159(Pt 6): 1010-1022, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23538715

RESUMO

Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.


Assuntos
Caulobacter crescentus/genética , Replicação do DNA , Plasmídeos , Ciclo Celular , DNA Bacteriano/metabolismo , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real
18.
Nucleic Acids Res ; 40(3): 1148-59, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21976729

RESUMO

DNA replication initiation proteins (Reps) are subjected to degradation by cellular proteases. We investigated how the formation of nucleoprotein complex, involving Rep and a protease, affects Rep degradation. All known Escherichia coli AAA+ cytosolic proteases and the replication initiation protein TrfA of the broad-host-range plasmid RK2 were used. Our results revealed that DNA influences the degradation process and that the observed effects are opposite and protease specific. In the case of ClpXP and ClpYQ proteases, DNA abolishes proteolysis, while in the case of ClpAP and Lon proteases it stimulates the process. ClpX and ClpY cannot interact with DNA-bound TrfA, while the ClpAP and Lon activities are enhanced by the formation of nucleoprotein complexes involving both the protease and TrfA. Lon has to interact with TrfA before contacting DNA, or this interaction can occur with TrfA already bound to DNA. The TrfA degradation by Lon can be carried out only on DNA. The absence of Lon results with higher stability of TrfA in the cell.


Assuntos
Proteases Dependentes de ATP/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Endopeptidase Clp/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Plasmídeos/genética , Protease La/metabolismo , Estrutura Quaternária de Proteína , Proteólise
19.
FEMS Microbiol Rev ; 36(2): 408-34, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22092310

RESUMO

Repeated sequences are commonly present in the sites for DNA replication initiation in bacterial, archaeal, and eukaryotic replicons. Those motifs are usually the binding places for replication initiation proteins or replication regulatory factors. In prokaryotic replication origins, the most abundant repeated sequences are DnaA boxes which are the binding sites for chromosomal replication initiation protein DnaA, iterons which bind plasmid or phage DNA replication initiators, defined motifs for site-specific DNA methylation, and 13-nucleotide-long motifs of a not too well-characterized function, which are present within a specific region of replication origin containing higher than average content of adenine and thymine residues. In this review, we specify methods allowing identification of a replication origin, basing on the localization of an AT-rich region and the arrangement of the origin's structural elements. We describe the regularity of the position and structure of the AT-rich regions in bacterial chromosomes and plasmids. The importance of 13-nucleotide-long repeats present at the AT-rich region, as well as other motifs overlapping them, was pointed out to be essential for DNA replication initiation including origin opening, helicase loading and replication complex assembly. We also summarize the role of AT-rich region repeated sequences for DNA replication regulation.


Assuntos
Bactérias/genética , Sequências Repetitivas de Ácido Nucleico , Origem de Replicação , Replicon , Sequência de Bases , Dados de Sequência Molecular
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