High Molecular Crowding in Repair Foci Surrounding DNA Breaks, Measured by Fluorescence Lifetime Imaging Microscopy.

The density of mammalian cells is determined primarily by the protein content. Local concentration of proteins in a cell is tightly controlled and varies between the cytoplasm, nucleoplasm, and nucleoli. We demonstrate that repair foci that are formed in response to DNA breaks are much more densely packed with proteins than the surrounding nucleoplasm. Using fluorescence lifetime imaging (FLIM), we demonstrated that the local concentration of all proteins (i.e., the residing and recruited ones) in double- and single-strand DNA repair foci can be even 2.2 times higher than that in the surrounding nucleoplasm, which brings them close to the achievable maximum concentration. The highest protein density is found in the center of a repair focus and gradually decreases with distance from the DNA lesion. We hypothesize that a microenvironment characterized by such a high protein concentration may facilitate the formation of protein condensates, resulting in the stabilization of repair complexes.