Fluorescence microscopy and the DNA damage response

Some of the main causes of cancer development are mutations in DNA caused by un- or misrepaired DNA damage. DNA can be damaged by many different sources, including ionizing radiation, UV-C light and certain endogenous metabolites. Cells respond to DNA damage with a variety of mechanisms, together known as the DNA damage response (DDR). The DDR consists of activation of cell cycle checkpoints initiated by phosphorylation of histone variant H2AX by ATM or ATR, a transcriptional response and DNA repair. We use fluorescence microscopy techniques to study the cellular response to DNA damage. For this purpose, DDR proteins are either visualized by immunofluorescence, or they are genetically tagged with GFP or a spectral variant of GFP to enable their visualization in living cells. To study the behavior of DDR proteins, we use a number of techniques to induce DNA damage in living cells, including ionizing radiation and UV lasers. Many DDR proteins accumulate in small nuclear foci in response to DNA damage induction. These foci are thought to be the locations where DNA repair takes place. Interestingly, there appear to be at least two different types of foci, each containing a subset of DDR proteins. The smaller foci consist of proteins directly involved in DNA repair, while the larger foci have a role in cell cycle signaling, which involves changes in chromatin structure. We are interested in these changes in chromatin structure following DNA damage induction and in the communication between the chromatin domain and the sites of DNA repair. Techniques we use to study these processes include the above-mentioned laser-assisted DNA damage induction, confocal microscopy photobleaching techniques and high-throughput siRNA screens.