FIGURE 2.

Schematic overview of methods to induce random DNA breaks in the genome using radiation (top left) or chemical agents (top right). The energy of radiation can be transferred directly to the DNA molecule or can ionize other molecules like water that will then attack the DNA. In addition to DNA breaks, radiation damage induces additional modifications on the DNA, represented as stars, pentagons or triangles. Examples of chemical induction of DSBs by the direct attack of DNA (using drugs such as neocarzinostatin) or indirectly by affecting specific proteins (Etoposide inhibits the topoisomerase cycle) are shown. Experimental approaches that can be coupled with these methods to induce DSBs are also represented in the bottom. From left to right, chromatin fractionation, to observe the accumulation of a protein on the cytoplasmic (Cyto), nucleoplasmic (Nuc), or chromatin (Chrom) fractions; Immunofluorescence, to visualize the formation of nuclear foci using specific antibodies; SMART, to measure the length of resected DNA; PFGE, to visualize the presence of pieces of broken chromatin (in the figure, S. cerevisiae chromosomes untreatated (U) or at different times upon irradiation); and comet assay, to study the appearance of breaks at the single-cell level. For details, see the main text.