Published in Cancer Detection and Prevention 2000; 24(Supplement 1).

Generation of Rev3-deficient mice to determine the function of mutagenic translesion synthesis in mammals

PP Van Sloun PhD, JG Jansen PhD, RJ Romeijn, N de Wind PhD, JC Eeken PhD

Dept Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Center, Leiden, The Netherlands,

The yeast Rev3 gene encodes the catalytic subunit of polymerase zeta which is involved in translesion synthesis, a process responsible for the majority of spontaneous and induced mutations in yeast. AIM: Analysis of the biological consequences of replication-blocking lesions present during S-phase in mammalian cells. METHODS: Cloning of mouse Rev3 cDNA (1). Disruption of Rev3 in ES cells and mice. Immunohistochemistry and TUNEL-assay. RESULTS: Using a conventional gene knock-out approach we established 3 Rev3 heterozygous mouse lines and heterozygous littermates were interbred. No live Rev3 mutant mice have been obtained from either line whereas heterozygous mice were born according to normal Mendelian frequency and appeared normal. No Rev3 mutant cell lines could be established either by targeting of the second allele in ES cells, de novo ES cell derivation from heterozygous crosses nor from primary embryonic fibroblasts, indicating that REV3 is essential for cellular survival. Rev3 mutant embryos die before 11.5 d.p.c. and show developmental retardation. The number of apoptotic cells is higher in Rev3 mutant embryos compared to wild-type littermates possibly as a result of p53-mediated apoptosis. CONCLUSION: Endogenous DNA damage may give rise to replication blocks that ultimately lead to embryonal death. Alternatively, REV3 may play a role in another pathway, indispensable for embryonic development. The finding that the REV7 protein binds not only REV3 but also MAD2 may point to a cross-talk between the translesion synthesis machinery and mitotic checkpoint proteins. 1) Van Sloun et al. Mutat Res; 433(2):109-16.

KEY WORDS: hypomutability, translesion synthesis, DNA replication.

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Paper presented at the International Symposium on Impact of Biotechnology on Cancer Diagnostic & Prognostic Indicators; Geneva, Switzerland; October 28 - 31, 2000; in the section on genetic instability.