COMPARISON OF INCORPORATION AND EXTENSION OF NUCLEOTIDES IN-VITRO OPPOSITE 8-HYDROXYGUANINE (7,8-DIHYDRO-8-OXOGUANINE) IN HOT-SPOTS OF THE C-HA-RAS GENE

DNA templates with 8-hydroxyguanine (7,8-dihydro-8-oxoguanine, oh(8)Gua) at a site corresponding to the first or second position of codon 12 of the c-Ha-ras gene were prepared, and the nucleotides inserted opposite the modified base were compared. The Klenow fragment (KF) of Escherichia coli DNA polymerase I inserted C opposite oh(8)Gua at both positions. Tag DNA polymerase incorporated C and A opposite oh(8)Gua, and the ratio of C to A was higher at the first position than at the second position. DNA polymerase a (pol alpha) inserted A and C at the first position, and A at the second position of codon 12, indicating that the ratio of C to A was higher at the first position. Moreover, we studied the extensions of bases paired with oh(8)Gua by DNA polymerases with or without 3'-5' exonuclease activity. G and T opposite oh(8)Gua were removed, and subsequently C was inserted by KF, We found that an oh(8)Gua:A pair was recognized by the exonuclease activity of the enzyme and that A was partially subsituted by C. On the other hand, pol alpha extended only C and A opposite oh(8)Gua. No difference was observed with oh(8)Gua at the two positions. These results indicate that the ratio of nucleotides incorporated opposite oh(8)Gua depends on the sequence context, while there is no particular difference in the extension of base pairs involving oh(8)Gua by DNA polymerases.