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
- Deoxyribonucleotides metabolism chemistry MeSH
- DNA-Directed DNA Polymerase * metabolism chemistry MeSH
- DNA * chemistry biosynthesis metabolism MeSH
- Cations * chemistry MeSH
- Polymerase Chain Reaction MeSH
- Purines chemistry biosynthesis MeSH
- Pyrimidines chemistry MeSH
- Publication type
- Journal Article 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.
- MeSH
- Cells drug effects metabolism MeSH
- Stress, Physiological * drug effects MeSH
- Humans MeSH
- Antineoplastic Agents chemistry pharmacology MeSH
- DNA Replication * drug effects MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review 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.
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
- Azides chemistry MeSH
- Click Chemistry MeSH
- Deoxyribonucleotides chemical synthesis chemistry metabolism MeSH
- DNA Primers biosynthesis genetics MeSH
- Endonucleases metabolism MeSH
- Fluorescent Dyes chemistry MeSH
- Molecular Structure MeSH
- Oligonucleotides biosynthesis MeSH
- Polymerase Chain Reaction * MeSH
- Nucleic Acid Amplification Techniques * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
... of Membrane Lipids and Steroids 715 -- DNA Replication, Recombination, and -- Repair 745 -- RNA Synthesis ... ... and Splicing 781 Protein Synthesis 813 The integration of Metabolism 845 The Control of Gene Expression ... ... 617 -- 22.4.2 Intermediates in Fatty Acid Synthesis Are -- Attached to an Acyl Carrier Protein 618 - ... ... 622 -- 22.4.8 Sources of NADPH for Fatty Acid Synthesis 623 -- 22.4.9 Fatty Acid Synthase Inhibitors ... ... Work by Inhibiting Protein -- Synthesis 838 -- 29.5.2 Diphtheria Toxin Blocks Protein Synthesis in - ...
5th ed. xvii, 974 s. : il., tab., grafy ; 32 cm
