Sequence-Directed Base Mispairing in Human Oncogenes

Abstract

The most frequently observed mutations in ras oncogenes in solid human tumors are GC3AT transitions at the 3 G residue of the GG doublet in codon 12 of these oncogenes. We had shown previously that mutagenesis by thymidine occurred with the same sequence specificity in mammalian cells, in that mutagenesis occurred preferentially at the 3 G of GG doublets. In this study, in vitro DNA synthesis experiments were carried out to assess the effect of local DNA sequence on base mispairing in order to determine the mechanism of sequence-directed mutagenesis by thymidine and its possible relationship to activating point mutations in N-, Ki-and Ha-ras oncogenes in solid human tumors. To avoid complicating the interpretation of the results because of the occurrence of mismatch repair as well as base misincorporation, the experiments were carried out in a repair-free environment with exonuclease-free Klenow polymerase. The results of these experiments showed that misincorporation of deoxyribosylthymine (dT) occurred with several-fold-greater efficiency opposite the 3 G compared to the 5 G of the GG doublet in codon 12 of human ras oncogenes. These results further demonstrated that the relative difference in the extent of dT misincorporation opposite the 3 G and the 5 G of GG doublets in codon 12 in the various ras oncogenes was affected by the base immediately upstream of the doublet. Within the GG doublet, it was seen that the 5 G and 3 G residues had an effect on the extent of dT misincorporation opposite each other. The 5 G was shown to have a stimulatory effect on dT misincorporation opposite the 3 G, while the 3 G was shown to have an inhibitory effect on dT misincorporation opposite the 5 G. Presumably, these mutual interactions within GG doublets are additive, such that the large differential in dT misincorporation observed between the 3 G and 5 G residues in GG doublets is the end result of the combined stimulatory and inhibitory effects within these doublets. Since the observed pattern of dT misincorporation within GG doublets corresponds to the most frequent mode of activation of ras oncogenes in solid human tumors, the results of these experiments suggest that sequence-directed dT misincorporation may be involved in the pattern of activation of human ras oncogenes, by causing GC3AT transitions preferentially at the 3 G of the GG doublet in codon 12 of these oncogenes.Carcinogenesis is generally considered to be the result of a multistep process involving several genetic changes. During studies on carcinogenesis in mammalian systems, and in the process of trying to determine the molecular basis of neoplasia, members of the ras family of oncogenes (N-, K-and Ha-ras) have been implicated (1, 3-5, 7, 19). Mutations in these genes have been observed in various naturally occurring human tumors as well as in carcinogen-induced animal tumors (1, 3-5, 7, 19), and the transforming ras genes have been shown to be mutant alleles of cellular ras genes (1, 3-5, 7, 19). It also has been shown that the ability of mammalian ras ...

Energetic basis for selective recognition of T{middle dot}G mismatched base pairs in DNA by imidazole-rich polyamides

“…In human bladder carcinoma, for example, a G´C to A´T transition at the 3¢G of the GG doublet in codon 12 of the Ha-ras and Ki-ras proto-oncogene converts them to oncogenes (7±9). A T´G mismatch is often introduced by spontaneous deamination of 5-methylcytosine and can arise from errors in replication (10). Structural analysis of DNAs containing T´G mismatches, indicates that the mismatches adopt a wobble conformation and structural perturbations are mainly in the vicinity of the mismatch (11,12).…”

Recognition of Tmiddle dotG mismatched base pairs in DNA by stacked imidazole-containing polyamides: surface plasmon resonance and circular dichroism studies

“…For example, in human bladder carcinoma a G•C→A•T transition at the 3′-G of the GG doublet in codon 12 of the Ha-ras and Ki-ras proto-oncogenes converts them to oncogenes (7)(8)(9). A C→T transition to give a T•G mismatch is often introduced by spontaneous deamination of 5-methylcytosine and can arise from errors in replication (10). Although ∼3% of cytosine residues in the human genome are methylated, mutations in 5-methylcytosine account for about one-third of the single base mutations that have been observed in inherited human diseases (10).…”

“…A C→T transition to give a T•G mismatch is often introduced by spontaneous deamination of 5-methylcytosine and can arise from errors in replication (10). Although ∼3% of cytosine residues in the human genome are methylated, mutations in 5-methylcytosine account for about one-third of the single base mutations that have been observed in inherited human diseases (10). While specific T•G mismatch repair systems exist in cells, some T•G sequences escape from being repaired (11).…”