Synchronous cell populations are commonly used for the analysis of various aspects of cellular metabolism at specific stages of the cell cycle. Cell synchronization at a chosen cell cycle stage is most frequently achieved by inhibition of specific metabolic pathway(s). In this respect, various protocols have been developed to synchronize cells in particular cell cycle stages. In this review, we provide an overview of the protocols for cell synchronization of mammalian cells based on the inhibition of synthesis of DNA building blocks-deoxynucleotides and/or inhibition of DNA synthesis. The mechanism of action, examples of their use, and advantages and disadvantages are described with the aim of providing a guide for the selection of suitable protocol for different studied situations.

• Klíčová slova
• DNA replication, S phase, cell cycle, deoxyribonucleotide triphosphates synthesis, ribonucleotide reductase, thymidine, thymidylate synthase,
• MeSH
• buněčné dělení * MeSH
• buněčný cyklus * MeSH
• DNA antagonisté a inhibitory   biosyntéza   MeSH
• lidé MeSH
• replikace DNA * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• DNA MeSH

Previous studies of polymerase synthesis of base-modified DNAs and their cleavage by restriction enzymes have mostly related only to 5-substituted pyrimidine and 7-substituted 7-deazaadenine nucleotides. Here we report the synthesis of a series of 7-substituted 7-deazaguanine 2'-deoxyribonucleoside 5'-O-triphosphates (dG(R) TPs), their use as substrates for polymerase synthesis of modified DNA and the influence of the modification on their cleavage by type II restriction endonucleases (REs). The dG(R) TPs were generally good substrates for polymerases but the PCR products could not be visualised on agarose gels by intercalator staining, due to fluorescence quenching. The presence of 7-substituted 7-deazaguanine residues in recognition sequences of REs in most cases completely blocked the cleavage.

• Klíčová slova
• DNA, nucleotides, polymerases, pyrrolopyrimidines,
• MeSH
• deoxyribonukleotidy chemie   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA biosyntéza   chemie   metabolismus   MeSH
• guanin analogy a deriváty   chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 7-deazaguanine MeSH Prohlížeč
• deoxyribonukleotidy MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• guanin MeSH

Earlier studies have established that the average speed of a replication fork is two to three times slower in early S-phase than in late S-phase and that the intracellular 2'-deoxyribonucleoside 5'-triphosphate pools grow during S-phase. In this study, the effect of the exogenous 2'-deoxyribonucleoside 5'-triphosphate (dNTP) supply on the average replication speed in a synchronised population of human HeLa cells was tested. The speed of replication fork movement was measured on extended DNA fibers labelled with 2'-deoxythymidine analogues 5-chloro-2'-deoxyuridine and 5-iodo-2'-deoxyuridine. We show that the introduction of exogenous dNTPs accelerates the replication process at the beginning of DNA synthesis only. In late S-phase, the administration of additional dNTPs has no effect on the speed of replication forks. The availability of 2'-deoxynucleotides seems to be a rate-limiting factor for DNA replication during early S-phase.

• MeSH
• deoxyribonukleotidy farmakologie   MeSH
• fluorescenční mikroskopie MeSH
• HeLa buňky MeSH
• kinetika MeSH
• lidé MeSH
• replikace DNA účinky léků   MeSH
• S fáze účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deoxyribonukleotidy MeSH

We designed and synthesized a set of four 2'-deoxyribonucleoside 5'-O-triphosphates (dNTPs) bearing cationic substituents (protonated amino, methylamino, dimethylamino and trimethylammonium groups) attached to position 5 of pyrimidines or position 7 of 7-deazapurines through hex-1-ynyl or propargyl linker. These cationic dNTPs were studied as substrates in enzymatic synthesis of modified and hypermodified DNA using KOD XL DNA polymerase. In primer extension (PEX), we successfully obtained DNA containing one, two, three, or (all) four modified nucleotides, each bearing a different cationic modification. The cationic dNTPs were somewhat worse substrates compared to previously studied dNTPs bearing hydrophobic or anionic modifications, but the polymerase was still able to synthesize sequences up to 73 modified nucleotides. We also successfully combined one cationic modification with one anionic and two hydrophobic modifications in PEX. In polymerase chain reaction (PCR), we observed exponential amplification only in the case of one cationic modification, while the combination of more cationic nucleotides gave either very low amplification or no PCR product. The hypermodified oligonucleotides prepared by PEX were successfully re-PCRed and sequenced by Sanger sequencing. Biophysical studies of hybridization, denaturation, and circular dichroism spectroscopy showed that the presence of cationic modifications increases the stability of duplexes.

• MeSH
• deoxyribonukleotidy metabolismus   chemie   MeSH
• DNA-dependentní DNA-polymerasy * metabolismus   chemie   MeSH
• DNA * chemie   biosyntéza   metabolismus   MeSH
• kationty * chemie   MeSH
• polymerázová řetězová reakce MeSH
• puriny chemie   biosyntéza   MeSH
• pyrimidiny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 7-deazapurine MeSH Prohlížeč
• deoxyribonukleotidy MeSH
• DNA-dependentní DNA-polymerasy * MeSH
• DNA * MeSH
• kationty * MeSH
• puriny MeSH
• pyrimidiny MeSH

Enzymatic synthesis of short (10-22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified deoxyribonucleoside triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR. Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides.

• MeSH
• azidy chemie   MeSH
• click chemie MeSH
• deoxyribonukleotidy chemická syntéza   chemie   metabolismus   MeSH
• DNA primery biosyntéza   genetika   MeSH
• endonukleasy metabolismus   MeSH
• fluorescenční barviva chemie   MeSH
• molekulární struktura MeSH
• oligonukleotidy biosyntéza   MeSH
• polymerázová řetězová reakce * MeSH
• techniky amplifikace nukleových kyselin * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azidy MeSH
• deoxyribonukleotidy MeSH
• DNA primery MeSH
• endonukleasy MeSH
• fluorescenční barviva MeSH
• oligonukleotidy MeSH

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses.

• Klíčová slova
• replication stress, aphidicolin, camptothecin, cancer, cisplatin, etoposide, hydroxyurea,
• MeSH
• buňky účinky léků   metabolismus   MeSH
• fyziologický stres * účinky léků   MeSH
• lidé MeSH
• protinádorové látky chemie   farmakologie   MeSH
• replikace DNA * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• protinádorové látky MeSH

The effect of cloturin on biosynthesis of DNA, RNA and proteins in both P388 and Ehrlich ascites carcinoma (EAC) cells have been studied in vitro. Biosynthesis of macromolecules indicated by the incorporation rate of [14C]adenine (DNA, RNA), [14C]thymidine (DNA), [14C]uridine (RNA) and [14C]valine (proteins) were studied for concentration (75 to 600 mumol/l) and time dependence. Cloturin inhibits incorporation of all 14C-precursors into the TCA-insoluble fraction of both types of cells in proportion to its concentration. The complete inhibition of 14C-precursors was reached at the highest concentrations of cloturin (300 and 600 mumol/l). The fact that incorporation of four precursors is inhibited suggests that the effect of cloturin lies at an underlying level of energy generation or transfer, rather than at specific reactions in the biosynthesis of DNA and proteins. The rate of DNA synthesis is rapidly affected by the lowering of the level of any of the four deoxyribonucleotide triphosphates. Interference with the generation of high-energy phosphate bonds is one of the mechanisms available for induction of nucleotide deficiency. A depletion of nucleotide pools can serve as an efficient tool to inhibit cellular growth and to induce cell death under some circumstances.

• MeSH
• adenin metabolismus   MeSH
• DNA nádorová biosyntéza   MeSH
• Ehrlichův tumor metabolismus   MeSH
• experimentální nádory metabolismus   MeSH
• hořčičné sloučeniny farmakologie   MeSH
• kinetika MeSH
• leukemie P388 metabolismus   MeSH
• merkaptopurin analogy a deriváty   farmakologie   MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• nádorové proteiny biosyntéza   MeSH
• protinádorové látky farmakologie   MeSH
• RNA nádorová biosyntéza   MeSH
• thymidin metabolismus   MeSH
• uridin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenin MeSH
• Cloturin MeSH Prohlížeč
• DNA nádorová MeSH
• hořčičné sloučeniny MeSH
• merkaptopurin MeSH
• nádorové proteiny MeSH
• protinádorové látky MeSH
• RNA nádorová MeSH
• thymidin MeSH
• uridin MeSH