The BrdU comet assay was performed to detect newly synthesized single strand DNA as previously described (42 (link)). Cells were labeled with 100 μM BrdU for 30 min. BrdU labeled cells were washed with PBS-, placed into fresh medium and incubated for an additional 30 min, 1 h or 2 h to mature replicating DNA strands. Labeled cells were collected, suspended in low-melting-point agarose and added to agarose-coated CometSlides. Slides were incubated in an alkaline lysis solution according to the manufacturer's protocol (Trevigen, 4250-050-K). Following electrophoresis, slides were neutralized by treatment with 0.4 M Tris–HCl, washed with PBS-, and immunostained. Incorporated BrdU substituted newly replicated DNA was detected with an anti-BrdU antibody (BD BioScience, 555627), followed by anti-mouse IgG Alexa 488 (Invitrogen, A-11029). DNA was counter-stained with DAPI and slides were dried according to the manufacturer's instructions (Trevigen, 4250-050-K). Digital images were acquired using a 20× objective lens on a BD pathway 855 microscope, which was controlled by AttoVision (Becton Dickinson). Images were analyzed by CometScore software (TriTeK).