8-Hydroxyadenine (7, 8-dihydro-8-Oxoadenine) induces misincorporation inin vitroDNA synthesis and mutations in NIH 3T3 cells

Abstract

An oligodeoxyribonucleotide containing 8-hydroxyadenine (OH8Ade) was chemically synthesized and single- and double-stranded c-Ha-ras gene fragments with OH8Ade at the second position of codon 61 were prepared. The single-stranded ras gene fragment was used as a template for in vitro DNA synthesis with the Klenow fragment of Escherichia coli DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse DNA polymerase alpha. The former two enzymes exclusively incorporated dTMP opposite OH8Ade. The DNA polymerases alpha and beta misinserted dGMP, and dAMP and dGMP, respectively. The c-Ha-ras gene was constructed using the double-stranded ras gene fragment containing OH8Ade and was transfected into NIH 3T3 cells. The gene with OH8Ade induced focus formation, indicating that OH8Ade elicited point mutations in cells. When c-Ha-ras genes present in transformed cells were analyzed, an A-->G transition and an A-->C transversion were detected. These results indicate that OH8Ade induced misincorporation in in vitro DNA synthesis and mutations in mammalian cells.

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.…”

“…Since this apparent base skipping is not observed in the OxoControl or 8-oxodG templates, it can not be the result of simple primer repositioning. This effect of 8-oxodA has not been observed in other experimental systems where deletions could readily be detected [11,13,28]. Several differences in the PCR components may explain this unexpected result.…”

“…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].…”

Size and topology of exogenous DNA as determinant factors of transgene transcription in mammalian cells

“…18,19 Cellular DNA repair systems 21,22 remove these lesions to prevent the mutations resulting from the formation of DNA lesions. [23][24][25][26] The binding of proteins involved in DNA repair may be another inhibitory factor and cause lower transcription of DNA recovered from the gel. Thus, we purified closed circular molecules and linearized DNAs in the same gel to compare the effect of the topology precisely.…”