DNA Fiber Analysis of Replication Dynamics

Cells were pulse-labelled sequentially with 50 μM CldU (20 min) and 250 μM IdU (20 min), harvested and resuspended in PBS (0.5 × 10⁶ cell/ml). 2 μl drops of cell suspension were placed on microscope slides and lysed with 0.5% SDS, 0.2 M Tris pH 7.4, 50 μM EDTA in 10 μl for 6 min at RT. Slides were tilted 15°C to spread DNA fibers, air-dried, fixed in –20°C methanol:acetic acid (3:1) for 2 min and stored at 4°C overnight. Slides were then incubated in 2.5 M HCl (30 min/RT) to denature DNA and washed (3×) in PBS. Blocking solution (1% bovine serum albumin in PBS, 0.1% Triton X-100) was added for 1 h at RT. Slides were incubated with anti-CldU, IdU and ssDNA primary antibodies for 1 h at RT, washed and incubated with the corresponding secondary antibodies for 30 min. Prolong mounting media (Invitrogen) was used. Images were acquired in a DM6000 B Leica microscope with an HCX PL APO 40×, 0.75 NA objective. Fork rate values were derived from the length of IdU tracks, measured using ImageJ software, and a conversion factor of 1 μm = 2.59 kb (36 (link)). >300 tracks were measured per condition. The percentage of origins activated during the first pulse (green-red-green tracks) was quantified relative to all replicative structures containing red signal. >500 total structures were scored in each case. Three biological replicates of each experiment were performed.