8-HYDROXYADENINE (7,8-DIHYDRO-8-OXOADENINE) INDUCES MISINCORPORATION IN IN VITRO DNA SYNTHESIS AND MUTATIONS IN NIH 3T3 CELLS

摘要

An oligodeoxyribonucleotide containing 8-hydroxyadenine (OH(8)Ade) was chemically synthesized and single- and double-stranded c-Ha-ras gene fragments with OH(8)Ade 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 coil DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse DNA polymerase alpha. The former two enzymes exclusively incorporated dTMP opposite OH(8)Ade. 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 OH(8)Ade and was transfected into NIH 3T3 cells. The gene with OH(8)Ade induced focus formation, indicating that OH(8)Ade 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 OH(8)Ade induced misincorporation in in vitro DNA synthesis and mutations in mammalian cells.