Measuring Chromosome Pairing During Homologous Recombination in Yeast
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The precise organization of the genome inside the cell nucleus is vital to many cell functions including gene expression, cell division, and DNA repair. Here we describe a method to measure pairing of DNA loci during homologous recombination (HR) at a site-specific double-strand break (DSB) in Saccharomyces cerevisiae. This method utilizes a chromosome tagging system in diploid yeast cells to visualize both the DNA at the break site and the homologous DNA that serves as a repair template. DNA repair products are confirmed in parallel by genomic blot. This visualization method provides insight into the physical contact that occurs between homologous loci during HR and correlates physical interaction with the timing of DNA repair.
Key wordsDNA repair Homologous recombination Homolog pairing Homology search Repair foci Rad52 Fluorescence microscopy Yeast
We especially thank Robert Reid as well as the entire Rothstein laboratory, Michael Smith, Ivana Sunjevaric, Olga Marte, Gaël Fortin, and Keerthana Chetlapalli for experimental feedback and suggestions. We also thank Judith Miné-Hattab and Michael Lisby for their work in the initial construction of the yeast strains used in this methods chapter. Lastly, we thank Lorraine Symington and Roberto Donnianni for suggestions and help with genomic blotting. This work was supported by National Institutes of Health grants T32 GM007088 (to F.J.J.), T32 CA009503 (to F.J.J. and E.E.B.), R35 GM118180-S1 (to F.J.J.), T32 GM008798 (to E.E.B.), TL1 TR001875 (to E.E.B.), and R35 GM118180 (to R.R.).
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