Nucleic Acids Research, 2003, Vol. 31, No. 17 4981-4988
© 2003 Oxford University Press
Ribozyme-mediated REV1 inhibition reduces the frequency of UV-induced mutations in the human HPRT gene
1 Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40202, USA and 2 Department of Medicine, Division of Medical Oncology and Hematology, J. G. Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202, USA
*To whom correspondence should be addressed at 221A Baxter Biomedical Research/Pharmacology, 570 S. Preston Street, University of Louisville Medical Center, Louisville, KY 40202, USA. Tel: +1 502 852 2564; Fax: +1 502 852 2492; Email: wgmcgregor{at}louisville.edu
In yeast, mutations induced by UV radiation are dependent on the function of the Rev1 gene product, a Y-family DNA polymerase that assists in translesion replication with potentially mutagenic consequences. Human REV1 has been cloned, but its role in mutagenesis and carcinogenesis remains obscure. To examine the role of REV1 in UV mutagenesis in human cells and to evaluate its potential as a therapeutic target to prevent such mutations, we developed a ribozyme that cleaves human REV1 mRNA in vitro. Stable expression of the ribozyme in human cells reduced the target REV1 mRNA up to 90%. We examined the cytotoxic and mutagenic response to UV of seven independent clones that had reduced levels of endogenous REV1 mRNA. In each case, the clonogenic survival after UV was not different from that of the parental cell strains. In contrast, the UV-induced mutant frequencies at the endogenous HPRT locus were reduced up to 75% in cells with reduced levels of REV1 mRNA. The data support the idea that targeting the mutagenic translesion DNA replication pathway can greatly reduce the frequency of induced mutations.
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