Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).

• MeSH
• arginin chemie   MeSH
• DNA chemická syntéza   chemie   MeSH
• histony chemie   MeSH
• ketony chemická syntéza   chemie   MeSH
• nádorový supresorový protein p53 chemie   MeSH
• peptidy chemie   MeSH
• proteiny chemie   MeSH
• reagencia zkříženě vázaná chemická syntéza   chemie   MeSH
• sérový albumin hovězí chemie   MeSH
• skot MeSH
• thiminnukleotidy chemická syntéza   chemie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Streptococcus pneumoniae is a Gram-positive bacterium that is a major agent of community-acquired bacterial pneumonia, meningitis and sepsis. Although the mismatch repair function of S. pneumoniae has been assigned to the hexA-hexB gene products, an enzyme capable of the direct elimination of noncanonical nucleotides from the cytoplasm has not been described for this bacterium. Our results show that Spr1057, a protein with previously unknown function, is involved in the inactivation of mutagenic pyrimidine nucleotides and was accordingly designated PynA (pyrimidine nucleotidase A). Biochemical assays confirmed the phosphatase activity of the recombinant enzyme and revealed its metal ion dependence for optimal enzyme activity. We demonstrated that PynA forms a homodimer with higher in vitro activity towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate than towards canonical thymidine monophosphate. Furthermore, we showed via in vivo assays that PynA protects cells against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine and prevents the incorporation of the potentially mutagenic 5-bromo-2'-deoxyuridine (5-BrdU) into DNA. Fluctuation analysis performed under S. pneumoniae exposure to 5-BrdU revealed that the pynA null strain accumulates random mutations with high frequency, resulting in a 30-fold increase in the mutation rate. The data support a model in which PynA, a protein conserved in other Gram-positive bacteria, functions as a house-cleaning enzyme by selectively eliminating noncanonical nucleotides and maintaining the purity of dNTP pools, similar to the YjjG protein described for Escherichia coli.

• MeSH
• 5'-nukleotidasa chemie   metabolismus   MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• deoxyuridin metabolismus   MeSH
• kationty metabolismus   MeSH
• mutační rychlost * MeSH
• Streptococcus pneumoniae enzymologie   genetika   MeSH
• substrátová specifita MeSH
• thymidinmonofosfát metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

2'-Deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2'-deoxythymidine 5'-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 μM EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations.

• MeSH
• buněčné linie MeSH
• cytotoxiny metabolismus   toxicita   MeSH
• deoxyuridin analogy a deriváty   metabolismus   toxicita   MeSH
• DNA biosyntéza   genetika   metabolismus   MeSH
• inhibitory enzymů metabolismus   toxicita   MeSH
• intracelulární prostor účinky léků   metabolismus   MeSH
• lidé MeSH
• poškození DNA * MeSH
• proliferace buněk účinky léků   MeSH
• replikace DNA účinky léků   MeSH
• S fáze účinky léků   MeSH
• tetrahydrofoláty biosyntéza   MeSH
• thymidin metabolismus   farmakologie   MeSH
• thymidinmonofosfát metabolismus   MeSH
• thymidylátsynthasa antagonisté a inhibitory   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• nehodgkinský lymfom enzymologie   krev   patologie   MeSH
• prognóza MeSH
• progrese nemoci enzymologie   MeSH
• protinádorové látky škodlivé účinky   MeSH
• thymidinkináza antagonisté a inhibitory   krev   MeSH
• thymidinmonofosfát biosyntéza   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• srovnávací studie MeSH

At a concentration of 0.5 to 3 mmol/l, ATP stimulates the activity of mitochondrial DNA polymerase of Neurospora crassa under the optimum reaction conditions; at higher concentrations, an inhibitory effect is observed. 4-Chloromercuribenzoate (1 mmol/L), a thiol inhibitor, decreases the enzyme activity two-fold, while N-ethylmalcimide (2 mmol/L) has no effect. Ethidium bromide (up to 10 mumol/L) and heparin (up to 0.4 micrograms/mL) reduce the activity by 60%. ddTTP does not affect the DNA polymerase reaction. The best in vitro template is the activated calf-thymus DNA.

• MeSH
• adenosintrifosfát farmakologie   MeSH
• aktivace enzymů účinky léků   MeSH
• dideoxynukleotidy MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• ethidium farmakologie   MeSH
• heparin farmakologie   MeSH
• inhibitory syntézy nukleových kyselin * MeSH
• mitochondrie enzymologie   MeSH
• Neurospora crassa účinky léků   enzymologie   MeSH
• sulfhydrylová reagencia farmakologie   MeSH
• thiminnukleotidy farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH