Maintaining the integrity of genetic information is essential for the survival of cells. Recent advances in cell biological and microscopy methodologies have complemented traditional genetic and biochemical approaches, and they now permit the observation of spatiotemporal aspects of damaged chromosomal loci. In one of these approaches, integrated LacO/TetO operator sequences can be used as binding sites to physically tether onto chromatin any protein of interest when genetically fused to the respective repressors (LacR/TetR). This methodology has been the basis of several models to probe the spatial dynamics of DNA repair in the eukaryotic nucleus and to visualize genomic loci in yeast, fly, nematodes, and in mammalian cells. Further applications are the induction of localized DNA damage by immobilizing endonucleases at different genome sites in vivo, the assessment of the hierarchy of protein interactions within repair complexes, and the activation of the DNA damage response (DDR) by the physical tethering of DSB-repair factors on chromatin in the absence of damage. We outline here a protocol for the quantification of DDR activation upon the prolonged immobilization of single repair factors on chromatin or upon tethering of the endonuclease FokI. The outlined protocol requires basic cell culture and microscopy skills and allows the tethering of any protein of interest within 2-3 days.
Keywords: 53BP1; ATM; Chromatin; DNA damage response; Imaging; Immunofluorescence; Lac operator; Lac repressor; Mre11; Tethering; γH2AX.