Fig. 3. Unscheduled DNA synthesis (UDS) occurs at RP-A foci induced by restriction endonuclease. (A) Double immunofluorescence with affinity-purified rabbit anti-RP-A70 antibodies (RPA) and with streptavidin conjugated to FITC in order to reveal incorporation of biotinylated dNTPs (dNTPs). Demembranated sperm heads (4000 heads) pretreated with 0.02 units of EcoRI (RE+HSS) or mock (HSS) are incubated for 1 hour at 23°C with HSS containing 20 µM biotinylated dATP and dUTP. Cumulative images representing 10-12 confocal sections for each fluorescence channel are shown. Most fluorescence foci owing to dNTP and RP-A signals co-localize as shown in the merged figure. It should be mentioned that, in some foci, there is an imbalance between the two color intensities, one being much higher than the other. Therefore, the yellow color in the merged figure is not that apparent in some co-localized foci (arrows). With mock pretreated sperm, the signal of the biotinylated dNTPs is barely visible. Bars, 5 µm. (B) Cesium chloride centrifugation gradient. Intact sperm heads were incubated with LSS extracts for 2 hours in the presence of [³²P]dATP and with BrdCTP (LSS-HL) or without BrdCTP (LSS-LL). EcoRI-pretreated sperm heads (0.02 U EcoRI per 4000 sperm heads) were incubated with HSS extracts for 2 hours in the presence of ³²P-dATP and BrdCTP (HSS RE). Equivalent amounts in sperm heads of LSS-HL and LSS-LL samples and five equivalents of HSS 43 fractions were taken from each gradient and their refractometric index as well as the total radioactivity measured. On the graph, the radioactivity expressed in arbitrary units (AU) is plotted for each fraction. The density of the fraction with the highest radioactivity value is written for each sample above the respective peaks.