Homologues of natural epigenetic pyrimidine nucleotides were designed and synthesized. They included 5-ethyl-, 5-propyl-, 5-(1-hydroxyethyl)-, 5-(1-hydroxypropyl)- and 5-acetyl- and 5-propionylcytosine and-uracil 2'-deoxyribonucleosides and their corresponding 5'-O-triphosphates (dNXTPs). The epimers of...
Five
2′-deoxyribonucleoside triphosphates (dNTPs) derived
from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine,
5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were
prepared and systematically studied as substrates for nine DNA polymerases
in competition with natural dNTPs by primer extension experiments.
The incorporation of these substrates was evaluated by a restriction
endonucleases cleavage-based assay and by a kinetic study of single
nucleotide extension. All of the modified pyrimidine dNTPs were good
substrates for the studied DNA polymerases that incorporated a significant
percentage of the modified nucleotides into DNA even in the presence
of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate
for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2′-deoxyuridine
triphosphate was not the best substrate for SPO1 DNA polymerase, which
naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage.
The results shed light onto the possibility of gene silencing through
recycling and random incorporation of epigenetic nucleotides and into
the replication of modified bacteriophage genomes.
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