Rad52 Protein Function Study By Fluorescence Tagging

The Rad52 protein plays a significant role in DNA lesions repair by homologous recombination in eukariotic cells. Human Rad52 function somewhat overlaps with BRCA2 and has a role in cell survival in the absence of BRCA1-BRCA2 mediated recombination. Additional Rad52 function analysis and intracellular localization studies are probably necessary. We present a method for Rad22 protein tagging, a SchizosaccharomycespombeRad52 homologue, byCrerecombinase-mediated cassette exchange (RMCE) using the versatile pAW8 plasmid. Rad22 protein was C-termini yEGFP tagged; the resulting strain was analyzed by fluorescence microscopy. The yEGFP signal was observed (Rad22 foci) for 7.5 mu M camptothecin, 0.005% methyl methanesulfonate, and 4 mMhydroxyurea treated cells. The RMCE method was efficient, and the presence of tagged Rad22 protein was confirmed by westernblot and fluorescence microscopy.