Figure 6.

BRCA2 mobility changes after DNA damage. (A) The percentage of additionally bound BRCA2 particles was determined by SPT analysis after induction of DNA damage: 2 and 5 h after exposure to 10 Gy IR, after 1 h treatment with 1 mM HU, and after 24 h treatment with 1 µg/ml MMC (from at least six fields, nine nuclei, and 457 individual tracks for each sample, well above 1,000 tracks for most conditions). In the absence of induced DNA damage, between 51 and 68% of the BRCA2 particles were bound. Three experimental replicates are shown for each treatment. (B) From all track segments, CDF curves were derived for the different DNA damage treatments (solid lines). Global fitting (broken lines) of the curves yielded three D_app components, with D₁ = 1.15 µm²/s, D₂ = 0.05 µm²/s, and D₃ = 0.003 µm²/s indicating mobility (D₁) and transient binding interactions (D₂ and D₃). The percentage for these different mobility contributions shows that after DNA damage induced by IR, HU, and MMC, more of the observed BRCA2 is transiently bound, manifested as an amplitude decrease of D₁ to 15%, 12%, and 13%, respectively, compared with the control condition (27%). (C) 2D difference histograms display mobility changes (yellow-red for increased frequency or blue for decreased frequency) after DNA damage indicating the shift to more immobile states compared with control (as shown in Fig. 4 A). In response to DNA damage, particles spend less time in the mobile state. Change in relative frequency is indicated by the colors defined on the right.