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DNA Damage and Cellular Stress Responses

Decreased Frequency and Highly Aberrant Spectrum of Ultraviolet-Induced Mutations in the hprt Gene of Mouse Fibroblasts Expressing Antisense RNA to DNA Polymerase ¹

¹ Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC;
² Department of Pharmacology and Toxicology and Molecular Targets Program, Department of Medicine (Hematology and Medical Oncology), J.G. Brown Cancer Center, University of Louisville, Louisville, KY; and
³ Department of Immunology, The Scripps Research Institute, La Jolla, CA

Requests for reprints: Marilyn Diaz, Laboratory of Molecular Genetics, P. O. Box 12233, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 or William Glenn McGregor, 221 Baxter Biomedical Research/Pharmacology, University of Louisville Medical Center, 570 S. Preston Street, Louisville, KY 40202. Phone: (502) 852-2564; Fax: (502) 852-2492. E-mail: wgmcgregor{at}louisville.edu

In the budding yeast Saccharomyces cerevisiae, DNA polymerase (pol ) is responsible for the great majority of mutations generated during error-prone translesion replication of DNA that contains UV-induced lesions. The catalytic subunit of pol is encoded by the Rev3 gene. The orthologue of Rev3 has been cloned from higher eukaryotic cells, including human, but its role in mutagenesis and carcinogenesis remains obscure. Investigation into the cellular function of pol has been hindered by the fact that Rev3 knockout mice do not survive beyond midgestation, and embryonic stem cells used to derive these mice are not genetically stable. We have generated a transgenic mouse that expresses antisense RNA transcripts to mRev3 endogeneous RNA. These mice are viable, have greatly reduced levels of Rev3 transcript, and have reduced levels of B cells and impaired development of high-affinity memory B cells. Here, we report that exposure of fibroblasts derived from these mice to UV resulted in a 4–5-fold reduction in mutant frequency at the hprt locus at every dose examined, and the mutation spectrum was highly aberrant compared with the control cells. In the control cells, 80% of the mutations were transitions and 75% of these arose from photoproducts in the putative leading strand template. Strikingly, in transgenic cells, most of the mutations were transversions and there was a complete loss of strand bias. This mutation spectrum is highly aberrant and is similar to that induced by UV in human xeroderma pigmentosum variant cells, which lack polymerase . These data indicate that most UV-induced mutations are dependent on DNA pol , a function that has been conserved from yeast to higher eukaryotic cells. However, in mammalian cells, other DNA polymerase(s) may accomplish error-prone translesion replication and are responsible for residual UV mutagenesis observed in the absence of pol . Further, these data support a central role for DNA polymerase in the error-free bypass of UV photoproducts. The antisense Rev3 mice should be a useful model to study mutagenic lesion bypass by pol in mammalian cells and to investigate the role this polymerase plays in carcinogenesis.

Key Words: DNA polymerase • cancer • mouse • antisense • mutagenesis

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