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)Gu a) 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 oppo site the modified base were compared. The Klenow fragment (KF) of Esch erichia coli DNA polymerase I inserted C opposite oh(8)Gua at both pos itions. 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 seco nd position. DNA polymerase a (pol alpha) inserted A and C at the firs t 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 stu died the extensions of bases paired with oh(8)Gua by DNA polymerases w ith or without 3'-5' exonuclease activity. G and T opposite oh(8)Gua w ere removed, and subsequently C was inserted by KF, We found that an o h(8)Gua:A pair was recognized by the exonuclease activity of the enzym e and that A was partially subsituted by C. On the other hand, pol alp ha 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 r atio of nucleotides incorporated opposite oh(8)Gua depends on the sequ ence context, while there is no particular difference in the extension of base pairs involving oh(8)Gua by DNA polymerases.