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

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 prepar ed. The single-stranded ras gene fragment was used as a template for i n vitro DNA synthesis with the Klenow fragment of Escherichia coil DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse D NA polymerase alpha. The former two enzymes exclusively incorporated d TMP opposite OH(8)Ade. The DNA polymerases alpha and beta misinserted dGMP, and dAMP and dGMP, respectively. The c-Ha-ras gene was construct ed 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 fo cus formation, indicating that OH(8)Ade elicited point mutations in ce lls. When c-Ha-ras genes present in transformed cells were analyzed, a n A-->G transition and an A-->C transversion were detected. These resu lts indicate that OH(8)Ade induced misincorporation in in vitro DNA sy nthesis and mutations in mammalian cells.