Ribozyme-mediated REV1L inhibition reduces the frequency of UV-induced mutations in the human HPRT gene

Mutagenesis by ultraviolet light (UV) in human cells has been shown to be dependent on a Y-family DNA polymerase encoded by the REV1L gene (S. cerevisiae, Rev1-like). We developed hammerhead ribozymes that were predicted to cleave the mRNA of this gene to investigate the molecular mechanisms of mutagenesis, as well as for potential therapeutic agents. In vitro time course studies showed prompt cleavage of REV1L RNA by the active ribozyme, but not by a catalytically inactive sequence that differed by only one base. We transfected two independent normal human fibroblast cell strains with inactive or active ribozymes and selected several drug-resistant clones that expressed the ribozymes, as shown by RT-PCR. Northern blot analysis indicated that REV1L mRNA had decreased in several of the clones. The clonogenic survival of UV-irradiated clones that expressed active or inactive ribozymes was the same as that of the parental cell strains. However, the UV-induced mutant frequency at the endogenous HPRT locus was reduced by 3-fold in clones that expressed the active ribozymes at each UV dose examined in both cell lines. These results were consistent between two different cell strains, indicating that the reduction in mutant frequency in response to the ribozyme is not cell line specific. These results are consistent with in vitro studies of homologous yeast proteins, which indicate that Rev1 is required for mutagenic bypass of UV-induced photoproducts by DNA polymerase ζ. These data indicate that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations, and we hypothesize that delivery of REV1L ribozymes to sun-exposed skin will reduce the incidence of UV-induced skin cancer.