8-HYDROXYGUANINE (7,8-DIHYDRO-8-OXOGUANINE) IN HOT-SPOTS OF THE C-HA-RAS GENE - EFFECTS OF SEQUENCE CONTEXTS ON MUTATION SPECTRA

We previously reported that 8-hydroxyguanine (7,8-dihydro-8-oxoguanine ) at the second position of codon 12 of the c-Ha-ras gene induces many types of mutations in NIH3T3 cells. In this study we incorporated the modified base into the first and second positions of codon 12 in the coding strand and into the first position of codon 61 in the non-codin g strand of the gene using a new 8-hydroxyguanine phosphoramidite as a building block during oligonucleotide synthesis. The ras genes with 8 -hydroxyguanine were transfected into NIH3T3 cells and the mutations i nduced were analyzed. 8-Hydroxyguanine residues at the first positions of codons 12 and 61 induced mutations to T at the modified sites almo st exclusively. On the other hand, the DNA lesion at the second positi on of codon 12 induced a G --> A transition in addition to a G --> T t ransversion, confirming our previous results. Mutations in 5'-flanking sites were observed with 8-hydroxyguanine at the second position of c odon 12 or the first position of codon 61. These results indicate that 8-hydroxyguanine in mammalian cells mainly induces a G --> T transver sion at the modified site, but that other types of mutations are also elicited.