Stable G-Quadruplex Structures of Oncogene Promoters Induce Potassium-Dependent Stops of Thermostable DNA Polymerase
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Amplification of GC-rich regions of genomic DNA is hindered either by high stability of DNA double helix or as a result of alternative structure formation by a guanine-rich DNA strand. Such potential G-quadruplex (G4) sequences are fairly common in promoters of the human genome. The efficiency of PCR amplification of promoter sequences for several human oncogenes (MYC, NRAS, TERT, KRAS, KIT) was studied. We demonstrate that the efficiency of DNA polymerase is reduced in the presence of potassium ions. The primer-extension technique localized DNA polymerase stops at the 3′-ends of potential quadruplex sequences. The structural and thermodynamic properties of short G-rich oligonucleotides corresponding to the stops of DNA polymerase were analyzed. These oligonucleotides formed stable parallel G4 in the presence of potassium ions. Correlation between the stability of G4 structure and efficiency of DNA polymerase stops was revealed. The results provide a method for detecting new G4 structures in extended genomic sequences and also clarify the mechanism of inhibition of DNA polymerase in G-rich regions of DNA.
KeywordsDNA G-quadruplex promoter sequences PCR DNA polymerase
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