MISINCORPORATION OF DAMP OPPOSITE 2-HYDROXYADENINE, AN OXIDATIVE FORMOF ADENINE

Nucleotide incorporation opposite an oxidative form of adenine, 2-hydr oxyadenine (2-OH-Ade) was investigated. When a primed template with 2- OH-Ade was treated with an exonuclease-deficient Klenow fragment of Es cherichia coli DNA polymerase I (KFexo-), recombinant rat DNA polymera se beta (pol beta) or calf thymus DNA polymerase alpha (pol alpha), in corporation of dTMP and dAMP was observed. In addition, KFexo- inserte d dGMP as well. A steady-state kinetic study indicated that the insert ion of dAMP and dTMP opposite the DNA lesion occurred with similar fre quency with KFexo- and pol beta. Insertion of dTMP opposite 2-OH-Ade w as favored to that of dAMP by pol alpha. Chain extension from the A . 2-OH-Ade pair is less favored than that from the T . 2-OH-Ade pair by all three DNA polymerases. Analysis of full-length products of in vitr o DNA synthesis showed that dTMP and dAMP were incorporated by DNA pol ymerases and that exonuclease-proficient and -deficient Klenow fragmen ts also inserted dGMP opposite 2-OH-Ade. These results suggest that fo rmation of 2-OH-Ade from A in DNA will induce A-->T and A-->C transver sions in cells.