DNA Damage Repair Kinetics Assay

Repair kinetic was estimated in 1d and 7d protonemata after bleomycin or MMS treatment, as previously described [19 (link)]. Tissue was either flash-frozen in liquid N₂ (repair t = 0) or allowed to recover in liquid BCDAT medium for the indicated repair times and then frozen. DSBs after bleomycin treatment were detected by a comet assay, using neutral N/N protocol, whereas DNA single-strand breaks (SSBs) after MMS treatment were detected with A/N protocol, which includes, after the lysis of nuclei, a treatment with alkali to reveal breaks by unwinding the DNA double helix, as described in [23 (link),24 (link)]. Comets were stained with SYBR Gold (Molecular Probes/Invitrogen, Eugene, USA), viewed in epifluorescence with a Nikon Eclipse 800 microscope, and evaluated by the LUCIA Comet cytogenetic software (LIM Inc., Prague, Czech Republic). The fraction of DNA in comet tails (% tail-DNA) was used as a measure of DNA damage. In each experiment, the % tail-DNA was measured at seven time points, namely 0, 3, 5, 10, 20, 60, and 180 min, after the treatment and in control tissue without treatment. Measurements obtained in three independent experiments, totaling at least 300 comets analyzed per experimental point were plotted as % of remaining damage and statistically analyzed by Student’s t-test. Time-course repair data were analyzed for two-phase exponential decay kinetics by Prism v.5 program (GraphPad Software Inc., San Diego, CA, USA).