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Keywords:

  • DNA double-strand breaks;
  • DNA repair nuclear foci;
  • Mre11 and Rad50 proteins;
  • immunofluorescence;
  • intensity correlation analysis;
  • correlated pixels

Abstract

Upon induction of DNA double-strand breaks (DSBs), Mre11 and Rad50 proteins of the Mre11 DNA repair complex accumulate at the sites of DSBs and form discrete nuclear foci. Precision in scoring of Mre11/Rad50-containing foci depends upon detection of those foci, some of which have a fluorescence staining intensity that is too close to the fluorescence staining intensity of the remaining Mre11 and Rad50 proteins that have not been incorporated into foci. Human U-1 melanoma cells in exponential growth were irradiated with various doses of X-rays (0-12 Gy) to induce the formation of repair foci. Four hours after irradiation, cells were simultaneously labeled for Mre11 and Rad50 proteins, using a two-color immunofluorescence staining technique. Laser scanning confocal microscopy was used to collect the composite images of randomly selected cell nuclei. Intensity correlation analysis (ICA) of equally intense fluorescence signals from Mre11 and Rad50 proteins was performed to obtain the regions with correlated pixels. ICA permitted enhanced detection of low level fluorescence of Mre11/Rad50 foci (“hidden” foci) that can be barely detected upon imaging of only one protein. For example, while imaging of only one protein (either Mre11 or Rad50) in the nucleus of a 6 Gy-irradiated cell revealed 9 foci, imaging of two proteins with ICA revealed 11 foci. ICA permitted an evaluation of the dose dependence of nuclear foci in cells irradiated with various doses of X-rays, with focus formation increasing up to a dose of 6 Gy. Our data accumulated using two-color immunofluorescence staining of Mre11 and Rad50 proteins and ICA of these two target proteins provide a basis for enhanced detection and accuracy in the scoring of DNA repair foci. © 2008 International Society for Advancement of Cytometry