Characterization of mammalian Rad51 in homologous recombination and DNA repair

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Malysewich, Michael J.
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University of Guelph

Mammalian Rad51 is an ATP-dependent DNA-binding and strand-exchange protein that plays an important role in the homologous recombination and double strand break repair pathways. Rad51 is the eukaryotic homologue of RecA and is highly conserved across all species with over 50% homology between yeast and humans. In the yeast, 'Saccharomyces cerevisiae', Rad51 mutation and gene knockout has revealed an important role for this protein in homologous recombination and response to DNA damaging agents. In mammalian cells, loss of Rad51 is lethal, although 'in vitro' approaches combined with transfection of wild-type and mutant forms of Rad51 into suitable recipient cell lines has confirmed the essential role of this protein in DNA damage and recombination responses. To further characterize Rad51 in murine hybridoma cell lines, a novel approach of using stable expression of anti-Rad51 siRNA was adopted to knockdown the expression of Rad51 in mouse hybridoma cells. Cell lines bearing a Rad51 deficiency display a reduced gene targeting efficiency and show sensitivity to both ionizing radiation and mitomycin C (MMC) treatments. In the knockdown cells, formation of Rad51 foci is not up-regulated after MMC exposure. The perturbation of HR functions through loss of wild-type Rad51 is further underscored by the reduced efficiency of targeted homologous recombination, by the reduced capacity to perform ectopic homologous recombination, and by a skewing in the pathway of intrachromosomal homologous recombination, from a normally conservative gene conversion pathway to an error-prone deletion pathway. Therefore, the conclusions of this thesis confirm and extend previous findings and provide support for the hypothesis of an essential role for Rad51 in mammalian DNA repair and recombination responses.

Mammalian Rad51, DNA repair, Homologous recombination, Strand-exchange protein, Characterization