Structure andin vitroreplication of DNA templates containing 7,8-dihydro-8-oxoadenine

Abstract

Adenine residues in DNA are oxidized under the action of ionizing radiation at the C-8 position to give 7,8-dihydro-8-oxoadenine. The formation of this lesion can be considered a cause of mutations and carcinogenesis. Oligodeoxyribonucleotides 39 and 47 bases long containing a single 7,8-dihydro-8-oxoadenine (8-hydroxyadenine) residue were synthesized by using nucleoside phosphoramidites. They were used as templates to study the copies obtained in vitro by the Klenow fragment and the thermostable Taq DNA polymerase. 7,8-Dihydro-8-oxoadenine does not block the replication and thymine is incorporated opposite the damage. The modifications of the DNA duplex conformation provoked by 7,8-dihydro-8-oxoadenine are minor. 1H-NMR spectroscopy shows that the duplex is in a B form, the sugar in a normal position in the helix and the modified base in the anti position. NMR confirms that 7,8-dihydro-8-oxoadenine exists predominantly in the keto form.

DNA damage reduces Taq DNA polymerase fidelity and PCR amplification efficiency

“…This is consistent with the preferential incorporation of dTMP opposite 8-oxodA observed for the Klenow fragment, Taq polymerase, and eukaryotic polymerases α and β [11][12][13]. However, in other less abundant amplicons derived from the OxodA1 template, we observed a one base deletion immediately after the modified base.…”

“…Bypass of 8-oxodA, 8-oxodG, and abasic lesions by several DNA polymerases has been extensively characterized [4,[10][11][12]15,26]. In addition to impacting the rates of nucleotide insertion and extension, these modified bases can pair with normal bases and increase the rate of mutagenesis.…”

“…incorporate dTMP in the presence of all four dNTPs [11][12][13]. When nucleotides are presented individually, these two enzymes insert dTMP and to a lesser extent dGMP which demonstrates that 8-oxodA has limited mutagenic potential [12].…”

“…Replication across these lesions by DNA polymerases results in the misincorporation of nucleotides and the establishment of mutations in daughter chromosomes [1][2][3][4][5]. The synthesis of oligonucleotides containing chemically defined bases permits the study of translesion synthesis and consequent misincorporation by a number of DNA polymerases [6][7][8][9][10][11][12][13][14][15][16]. 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) can assume either a syn or anti-orientation depending on its base pairing partner [6][7][8][9].…”

“…DNA Polymerase I, the Klenow fragment, and polymerase β preferentially incorporate dCMP, while eukaryotic polymerases α and δ incorporate dAMP more frequently [10]. 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxodA) resides in the anti orientation and causes only minor conformational changes to duplexed DNA [11]. On synthetic templates containing 8-oxodA, the Klenow fragment of E. coli DNA polymerase I and Taq DNA polymerase exclusively *To whom correspondence should be addressed.…”