Immunofluorescence and microscopy

For γH2AX and RAD51 immunofluorescence, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and blocked with 1% normal goat serum (for γH2AX) or 3% BSA (for RAD51) in PBS for 1 h. For TOP1cc staining, cells were fixed with 4% paraformaldehyde for 15 min at 4 °C, permeabilized with 0.25% Triton X-100 in PBS for 15 min at 4 °C and treated with 1% SDS in PBS for 5 min at room temperature. Cells were washed five times with wash buffer (0.1% Triton X-100, 0.1% BSA in PBS) and blocked with 10% milk in 10 mM Tris-HCl pH 7.4 and 150 mM NaCl. Primary antibodies used were mouse anti-phospho-Histone H2A.X (Ser139) (Millipore, 05-636, 1:500), rabbit anti-RAD51 (Calbiochem, PC130, 1:300), and mouse anti-TOP1cc antibody⁵³ (1:100). Secondary antibodies used were goat anti-mouse IgG Alexa fluor 488 (Invitrogen, A11029, 1:2000) and goat anti-rabbit IgG Alexa fluor 488 (Invitrogen, A11034, 1:2000). Images were captured using a Zeiss Axio Scope.A1 fluorescent microscope equipped with a CCD camera (Jenoptik). Foci were scored using ImageJ on images captured with a 63× objective (for γH2AX) or counted manually (for RAD51 and TOP1cc). At least 100 cells were scored for focus formation by blinded observers.