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

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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 Escherichla coli DNA polymerase I, Taq DNA polymerase, rat DNA polymerase β and mouse DNA polymerase α. The former two enzymes exclusively incorporated dTMP opposite 0H8Ade. The DNA polymerases a and p 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.

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