title: Rad52 protein function study by fluorescence tagging.
abstract: 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 Schizosaccharomyces pombe Rad52 homologue, by Crerecombinase-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 microM camptothecin, 0.005% methyl methanesulfonate, and 4 mM hydroxyurea treated cells. The RMCE method was efficient, and the presence of tagged Rad22 protein was confirmed by Western-Blot and fluorescence microscopy.
authors: Faculty of Medicine, University of Medicine and Pharmacy "Grigore T. Popa" - Iasi.
authors: Gabriela
authors: G
authors: Bordeianu
authors: Elena
authors: E
authors: Petrescu-Danila
authors: Didona
authors: D
authors: Ungureanu
authors: B
authors: B
authors: Stoica
authors: Raluca
authors: R
authors: Stanescu
authors: A
authors: A
authors: Cernomaz
authors: M
authors: M
authors: Rusu
mesh_terms: D015153:Blotting, Western; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics* / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D051721:Rad52 DNA Repair and Recombination Protein; D029702:Schizosaccharomyces pombe Proteins; C485184:enhanced green fluorescent protein; C085412:rad52 protein, S pombe; D049452:Green Fluorescent Proteins