Quantitative analysis of nuclear deformations and DNA damage foci dynamics by live-cell imaging

The correct repair of DNA Double Strand Breaks (DSBs) is fundamental to prevent the loss of genetic information, mutations, and chromosome rearrangements. An emerging determinant of DNA repair is chromatin mobility. However, how chromatin mobility can influence DSBs repair is still poorly understood. While increased mobility is generally associated with the correct repair by Homologous Recombination (HR) of DSBs generated in heterochromatin, it promotes the mis-repair of multiple distal DSBs by Non-Homologous End Joining (NHEJ). Here we describe a method for detecting and quantifying DSBs mobility by live-cell imaging in the context of multiple DSBs prone to mis-repair by NHEJ. In addition, we discuss a set of parameters that can be used for quantitative and qualitative analysis of nuclear deformations and to discard nuclei where the deformation could affect the analysis of DSBs mobility. While this method is based on the visualization of DSBs with the mCherry-53BP1-2 fusion protein, we believe that it can also be used to analyze the mobility of nuclear foci formed by different fluorescent proteins.