Dissection of the recombination functions of Saccharomyces cerevisiae Rad52

Homologous Recombination (HR) is an essential DNA repair pathway used by cells to combat DNA damage and maintain genome integrity. Faithful replication of lost or damaged sequence is ensured in HR, where a sister homologue is used to guide repair. Central to this HR process is the Rad51 recombinase, a protein responsible for the formation of an active presynaptic nucleoprotein filament that is able to conduct homology search in the homologous duplex. In order to facilitate filament formation, recombination mediators are required to load Rad51 onto the DNA tails. The most commonly studied mediator protein is Rad52. In Saccharomyces cerevisiae, Rad52 plays two distinct roles that are germane to DNA repair: it is responsible for mediating the assembly of Rad51 onto single-strand DNA (ssDNA) to allow HR to proceed; and is also required for the annealing of complementary ssDNA that is complexed with the ssDNA-binding protein RPA. Work presented herein describes a detailed biochemical characterization of Rad52 to elucidate how each of its specific activities contributes to its overall function during HR. In Chapter 2, the significance of the Rad52-RPA interaction on the formation of repair centers within the cell, as well as the effect of this interaction on the ability of Rad52 to carry out its mediator function is addressed. The annealing function of Rad52 is tackled in Chapter 3, where I show that the evolutionary-conserved amino terminal DNA binding domain of Rad52 is crucial for this activity. The knowledge gained from this research will hopefully be useful in guiding future studies on the human recombination mediator BRCA2, in which a compromised activity gives rise to cancer phenotypes in humans.