8-Hydroxyguanine in a mutational hotspot of the c-Ha-ras gene causes misreplication, 'action-at-a-distance' mutagenesis and inhibition of replication.

Mutations in particular codons of c-Ha-ras have a strong activating potential, and an activated ras oncogene has been found in a number of human cancers. Using fragments of the human c-Ha-ras gene containing 8-hydroxyguanine (8-OH-G) in codon 12, we provide evidence for highly complex biochemical events leading to activation of the oncogene. Replication with DNA polymerases alpha (Pol(alpha)) and beta (Pol(beta)) led to misincorporation of dAMP, while DNA polymerase eta (Pol(eta)) caused additional insertion of dGMP. For the first time we report an 'action-at-a-distance' mutagenic effect for Pol(eta). Replication catalyzed by this enzyme resulted in misincorporating dAMP, dTMP and dGMP opposite non-oxidized guanine 3'-flanked by 8-OH-G. Interestingly, two adjacent 8-OH-G residues greatly relaxed the specificity of Pol(eta), which in this system was able to incorporate all four nucleotides. Moreover, two adjacent 8-OH-G residues completely blocked Pol(alpha) and strongly inhibited Pol(beta), whereas Pol(eta) was entirely resistant to this inhibition. These results suggest an important role for Pol(eta) in inducing hypermutability in codon 12. Our observations are important for understanding the consequences of 8-OH-G being positioned within the mutational hot spots of oncogenes, the outcome of which appears to be relatively complex even in minimal in vitro systems.