2.6. DNA Fiber Assay

Exponentially growing cells were pulse labeled with 25 μM CldU (Sigma) followed by 250 μM IdU (Sigma) for 30 min each. HU was given for 2 h between both labels, Inhibitors 2 h before labelling, MMC was given at the beginning of the 2nd pulse labelling time. Labeled cells were harvested, DNA fiber spreads prepared and stained as described [27]. Fibers were examined using an Axioplan 2 fluorescence microscope (Zeiss, Oberkochen, Germany). CldU and IdU tracks were measured using ImageJ (version 1.48, Company, City, State abbrev. If USA, Country) [27,28]. At least 300 forks were analyzed.