Projekt je zaměřen na studium bázových excizních oprav (BER) na buněčné i molekulární úrovni ve vztahu k strukturní a funkční organizaci aktivních replikonů a na cílené poškození BER s cílem nalézt nové možnosti léčby rakoviny. Očekáváme, že interakce enzymů zapojených v BER s modelovými duplexy DNA modifikovanými v kritických místech strukturně odlišnými nukleotidovými jednotkami (nukleosidové analogy a/nebo fosfonátové typy internukleotidových vazeb) umožní získání poznatků nutných pro cílené poškození BER systému. Projekt je navržen s cílem pokrýt celou dráhu od navržení a syntézy nukleosidových, nukleotidových a oligonukleotidových analogů, přes analýzu nukleosidových a nukleotidových koncentrací a mikroskopické analýzy DNA replikace a replikačních aparátů až po molekulární analýzu izolovaných DNA/proteinových komplexů.; The project is focused on the study of base excision repair (BER) at both cellular and molecular levels with respect to the structural and functional organization of active replicons and the targeted impairment of BER with the aim to find new possibilities of cancer treatment. We anticipate that interactions of BER enzymes with the model DNA duplexes modified in critical sites with the structurally diverse unnatural nucleotide units (nucleoside analogs and/or phosphonate types of the internucleotide linkages) enable the achievement of fundamental knowledge on the targeted impairment of base excision repair. The project is designed with the aim of covering the whole pathway from the design and synthesis of nucleoside, nucleotide, and oligonucleotide analogues, through the analysis of nucleoside and nucleotide pools, microscopy analysis of DNA replication and repair machineries up to the molecular analysis of isolated protein-DNA complexes.

• MeSH
• DNA-glykosylasy MeSH
• hmotnostní spektrometrie MeSH
• mikroskopie MeSH
• nádory terapie   MeSH
• nukleosidy MeSH
• oprava DNA genetika   MeSH
• organofosfonáty MeSH
• poruchy opravy DNA genetika   MeSH
• replikace DNA MeSH
• replikon MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• genetika, lékařská genetika
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.

• MeSH
• časové faktory MeSH
• chromozomální nestabilita MeSH
• cyklin-dependentní kinasy metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA biosyntéza   genetika   MeSH
• endodeoxyribonukleasy metabolismus   MeSH
• endonukleasy genetika   metabolismus   MeSH
• fosforylace MeSH
• fragilní místa na chromozomu * MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• helikasy RecQ genetika   metabolismus   MeSH
• lidé MeSH
• mitóza * MeSH
• oprava DNA * MeSH
• poškození DNA MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikační počátek * MeSH
• RNA interference MeSH
• segregace chromozomů MeSH
• transfekce MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51(T131P) mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.

• MeSH
• časové faktory MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• DNA-polymerasa I metabolismus   MeSH
• DNA-polymerasa III metabolismus   MeSH
• DNA biosyntéza   genetika   MeSH
• endodeoxyribonukleasy genetika   metabolismus   MeSH
• exodeoxyribonukleasy genetika   metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• nestabilita genomu MeSH
• protein BRCA2 genetika   metabolismus   MeSH
• proteiny Xenopus genetika   metabolismus   MeSH
• rekombinasa Rad51 genetika   metabolismus   MeSH
• replikace DNA * MeSH
• replikační počátek MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Xenopus laevis genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants are able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing.

• MeSH
• apoptóza MeSH
• chromatin genetika   MeSH
• helikasy RecQ genetika   metabolismus   MeSH
• imunoenzymatické techniky MeSH
• imunoprecipitace MeSH
• interakční proteinové domény a motivy MeSH
• kur domácí genetika   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• MCM komplex, komponenta 2 genetika   metabolismus   MeSH
• MCM komplex, komponenta 7 genetika   metabolismus   MeSH
• MCM proteiny genetika   metabolismus   MeSH
• messenger RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• nádorové buňky kultivované MeSH
• nádory kostí genetika   metabolismus   patologie   MeSH
• osteosarkom genetika   metabolismus   patologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• povrchová plasmonová rezonance MeSH
• proliferace buněk MeSH
• průtoková cytometrie MeSH
• replikace DNA * MeSH
• replikační počátek genetika   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Cdc7 (cell division cycle 7) kinase together with its activation subunit ASK (also known as Dbf4) play pivotal roles in DNA replication and contribute also to other aspects of DNA metabolism such as DNA repair and recombination. While the biological significance of Cdc7 is widely appreciated, the molecular mechanisms through which Cdc7 kinase regulates these various DNA transactions remain largely obscure, including the role of Cdc7-ASK/Dbf4 under replication stress, a condition associated with diverse (patho)physiological scenarios. In this review, we first highlight the recent findings on a novel pathway that regulates the stability of the human Cdc7-ASK/Dbf4 complex under replication stress, its interplay with ATR-Chk1 signaling, and significance in the RAD18-dependent DNA damage bypass pathway. We also consider Cdc7 function in a broader context, considering both physiological conditions and pathologies associated with enhanced replication stress, particularly oncogenic transformation and tumorigenesis. Furthermore, we integrate the emerging evidence and propose a concept of Cdc7-ASK/Dbf4 contributing to genome integrity maintenance, through interplay with RAD18 that can serve as a molecular switch to dictate DNA repair pathway choice. Finally, we discuss the possibility of targeting Cdc7, particularly in the context of the Cdc7/RAD18-dependent translesion synthesis, as a potential innovative strategy for treatment of cancer.

• MeSH
• chromatin metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• fyziologický stres * MeSH
• lidé MeSH
• nádory metabolismus   patologie   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• replikace DNA * MeSH
• replikační počátek MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

A derivative of Azospirillum brasilense Sp245, Sp245.5, which spontaneously lost 85 and 120 MDa replicons upon the formation of a new megaplasmid, has been shown to produce a novel lipopolysaccharide and to lose Calcofluor-binding polysaccharides. As compared to Sp245, the derivative displays notably increased heavy metal tolerance. The phenotypes of Sp245 and Sp245.5 are characterized by the following minimal inhibitory concentrations (MICs) of heavy metals: 0.5 and 0.9 μmol l(-1) of Ag(+), 0.4 and 0.7 mmol l(-1) of Co(2+), 0.9 and 4.7 mmol l(-1) of Cu(2+), and 3.1 and 11.5 mmol l(-1) of Zn(2+), respectively. In Sp245, in the presence of a nonlethal concentration (0.625 μmol l(-1)) of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), the MIC of cobalt, copper, and zinc drop 1.3- to 1.6-fold, but the low tolerance to silver is unaffected. In Sp245.5, CCCP does not affect cobalt tolerance, suppresses tolerance to copper and silver to the wild-type levels, and causes a 1.4-fold decrease in resistance to zinc. Therefore, significant elevation of heavy metal tolerance in Sp245.5 seems caused by the induction/overexpression of the proton-dependent efflux of certain metal ions. The novel cell surface and other unknown factors could also be responsible for the increased tolerance of A. brasilense Sp245.5 to heavy metals.

• MeSH
• Azospirillum brasilense genetika   metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• genová přestavba MeSH
• plazmidy genetika   MeSH
• replikon MeSH
• rostliny mikrobiologie   MeSH
• těžké kovy metabolismus   MeSH
• vývoj rostlin MeSH
• Publikační typ
• časopisecké články MeSH

The essential role of MCM 2–7 proteins in the initiation of DNA replication in all eukaryotes is well known. Their role in replication elongation is supported by numerous studies, but there is still a knowledge gap in this respect. Even though biochemical studies have established an association of MCM proteins with replication forks, previous immunofluorescence studies in mammalian cells have suggested that MCM 2–7 proteins are displaced after replication initiation from sites of DNA replication. Therefore, we used a robust statistical method to more precisely analyse immunofluorescence localization of MCM 2 proteins with respect to the DNA replication foci. We show that despite the predominantly different localization of MCM 2 and replication signals, there is still a small but significant fraction of MCM 2 proteins that co-localize with DNA replication foci during most of S phase. The fluorescence localization of the MCM 2 proteins and DNA replication may thus reflect an active function of MCM 2 proteins associated with the replication foci and partially explain one facet of the “MCM paradox”.

• MeSH
• buněčné jádro metabolismus   MeSH
• chromatin metabolismus   MeSH
• financování organizované MeSH
• fluorescenční protilátková technika MeSH
• HeLa buňky MeSH
• jaderné proteiny metabolismus   MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• neparametrická statistika MeSH
• počítačové zpracování obrazu metody   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• replikační počátek MeSH
• S fáze MeSH
• Check Tag
• lidé MeSH

According to a general paradigm, proper DNA duplication from each replication origin is ensured by two protein complexes termed replisomes. In prokaryotes and in budding yeast Saccharomyces cerevisiae, these two replisomes seem to be associated with one another until DNA replication initiated from the origin has finished. This arrangement results in the formation of the loop of newly synthesized DNA. However, arrangement of replisomes in other eukaryotic organisms including vertebrate cells is largely unknown. Here, we used in vivo labeling of DNA segments in combination with the electron microscopy tomography to describe the organization of replisomes in human HeLa cells. The experiments were devised in order to distinguish between a model of independent replisomes and a model of replisome couples. The comparative analysis of short segments of replicons labeled in pulse-chase experiments of various length shows that replisomes in HeLa cells are organized into the couples during DNA replication. Moreover, our data enabled to suggest a new model of the organization of replicated DNA. According to this model, replisome couples produce loop with the associated arms in the form of four tightly associated 30nm fibers.

• MeSH
• bromodeoxyuridin metabolismus   MeSH
• buněčné jádro metabolismus   ultrastruktura   MeSH
• chromatin fyziologie   ultrastruktura   MeSH
• deoxyuracilnukleotidy metabolismus   MeSH
• DNA-dependentní DNA-polymerasy chemie   metabolismus   MeSH
• financování organizované MeSH
• HeLa buňky MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• modely genetické MeSH
• multienzymové komplexy chemie   metabolismus   MeSH
• počítačové zpracování obrazu MeSH
• replikace DNA fyziologie   MeSH
• replikon genetika   MeSH
• tomografie elektronová MeSH
• Check Tag
• lidé MeSH

OBJECTIVES: In this study, we analysed field isolates of Salmonella enterica serovar Typhimurium for the presence of conjugative plasmids transferring resistances to antibiotics. METHODS: Altogether 23 strains were analysed for the presence of conjugative R-plasmids. In the case of successful conjugation, the R-plasmids were characterized by PCR for antibiotic resistance genes, integrons and replicon typing. Variable regions of integrons were sequenced. RESULTS: Conjugation and transfer of antibiotic resistance was observed in 12 strains. Conjugative plasmids in these strains belonged to the IncI1 and IncHI1 replicons and four of them transferred antibiotic resistance associated with class I integrons. In two cases, resistance to tetracycline and/or ampicillin was not transferred by conjugation to approximately 10% of the transconjugants. Detailed characterization showed that the loss of both resistances was associated with the loss of Tn3 (bla(TEM)) and Tn1721 [tet(A)] from the conjugative plasmids p9046 and p9134. However, when only the tetracycline resistance was lost, the Tn1721 was replaced with a partial sequence of rck, and with complete coding sequences of srgA, srgB, ORF7 and pefI originating from the Salmonella Typhimurium virulence plasmid. CONCLUSIONS: Two plasmids from our collection were capable of recombination with the virulence plasmid of Salmonella Typhimurium and subsequently spread both antibiotic resistance and virulence genes to the recipient.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence MeSH
• DNA bakterií genetika   MeSH
• faktory virulence genetika   MeSH
• financování organizované MeSH
• genotyp MeSH
• genová přestavba MeSH
• integrony MeSH
• konjugace genetická MeSH
• lidé MeSH
• plazmidy MeSH
• polymerázová řetězová reakce MeSH
• pořadí genů MeSH
• rekombinace genetická MeSH
• replikon MeSH
• Salmonella typhimurium genetika   izolace a purifikace   patogenita   účinky léků   MeSH
• syntenie MeSH
• transpozibilní elementy DNA MeSH
• virulence MeSH
• Check Tag
• lidé MeSH

• MeSH
• Acetobacterium genetika   metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• DNA bakterií genetika   MeSH
• molekulární sekvence - údaje MeSH
• plazmidy genetika   MeSH
• replikon genetika   MeSH
• sekvenční homologie MeSH