Fig. 4. The Xenopus homolog of Ku86 localizes in foci on restriction-enzyme-treated sperm pronuclei that largely coincide with RP-A foci and with the sites of UDS. (A) Immunofluorescence with purified anti-XKu86 antibodies on untreated (HSS) and restriction-enzyme-treated (HSS+RE) sperm heads incubated in HSS. The restriction enzyme treatment induced an accumulation of XKu86 in multiple bright foci. Double immunofluorescence with affinity-purified rabbit anti-Xenopus Ku86 antibodies (Anti-XKu86) and chicken anti-RP-A70 (RP-A) antibodies on sperm heads (
4000 heads) pre-treated with 0.05 units of EcoRI (Sperm+RE) and incubated with HSS for 1 hour at 23°C. Most fluorescence RP-A and Ku foci co-localize. Owing to an imbalance between the two color intensities, the yellow color in the merged figure is not that apparent in some co-localized foci (arrows) (the same argument applies to C). (C) Double immunofluorescence with affinity-purified rabbit anti-XKu86 antibodies (XKu86) and with streptavidin conjugated to FITC to visualize the incorporation of biotinylated dNTPs (dNTPs). EcoRI-pretreated sperm were incubated with HSS in the presence of 20 µM biotinylated dATP and dUTP for 1 hour at 23°C. In the merged figure, most fluorescence foci co-localize. Cumulative images representing 10-12 confocal sections for each fluorescence channel are shown. Bars, 3 µm.
