Although the removal and repair of DNA damage is essential for the survival of cells and organisms, all cells can complete DNA replication in the presence of some unrepaired DNA damage. A specialized group of newly recognized DNA polymerases assist in accomplishing replication of damaged genomes. These enzymes insert nucleotides opposite lesions that normally block eplication forks. The process helps cells survive genotoxic insults from both endogenous and environmental sources, but often introduces incorrect bases and is mutagenic. Accumulation of mutations can lead to neoplastic transformation of a cell and cancer in an organism. A key enzyme in forming mutations during translesion synthesis is DNA polymerase zeta (zeta). The long-term objectives of this research are to understand how mammalian Pol zeta contributes to survival of the organism and to genetic stability in relation to cancer. Disruption of the mouse Rev3L gene, encoding the catalytic subunit of Pol zeta, leads to lethality midway through embryonic gestation. It is likely that Pol zeta is essential for development because embryos follow a strictly timed program of rapid division that relies on effective tolerance of DNA lesions by a Pol zeta-dependent process. The research in this proposal will use methods that allow analysis of Pol zeta function in cultured and adult cells.
The specific aims of this project:
Aim 1 is to test whether the absence of Pol zeta in mammalian cells is associated with hypersensitivity to DNA damage, impaired viability and genome instability. We hypothesize that a lack of Pol zeta during development causes a loss of viability as rapidly dividing cells accumulates blocked DNA replication forks and DNA breaks. We have isolated a mouse cell line with a genetic disruption of Rev3L, making it now possible to test these predictions. Rev3L (-I-) cells will be analyzed for impaired growth, viability, and progression through the cell cycle, sensitivity to a range of DNA damaging agents, gross chromosomal arrangements by spectral karyotyping, and frequency of sister chromatid exchange. The results will indicate the most important functions of mammalian Pol zeta with respect to DNA lesions and repair pathways.
Aim 2 is to determine which tissues are most dependent on Rev3L for normal development and function, which will point towards the most important biological roles of the enzyme. Pol zeta will be lowered and eliminated in cells of a mouse using conditional gene disruption technology. A construct having key exons of Rev3L flanked by LoxP sites will be used to disrupt the gene in adult tissues or at specific times during the lifespan of the animal. It is predicted that the tissues most dependent on Pol zeta function will be those most rapidly proliferating, or with the highest exposure to oxygen free- radicals or other DNA damaging agents.
Aim 3 is to test the hypothesis that animals with cell types, which can survive in the absence of Pol z will have an altered incidence of spontaneous, and DNA damage-induced tumorigenesis. If there is more chromosomal instability in Pol zeta (-I-) cells as indicated by our preliminary studies, it is predicted that tumor incidence will be increased. Using mosaic knockout mice and mice with tissue-specific disruptions of Pol zeta, experiments will be initiated to measure the incidence of spontaneous and induced tumors.
