DNA repair in plants studied by comet assay

Comet assay in plants.From the first description of the comet assay with isolated nuclei rather than whole cells it became evident that assay is well suited to be applied in plants (Koppen & Verschaeve, 1996). Disintegration of tissue by quick chopping with a razor blade, direct collection of released nuclei by patting and pipetting enables to process samples in time shorter than 2 minutes, the time prerequisite to study quick repair (Kozak et al, 2009). Plants are due to their sessile nature permanently exposed to environmental stresses (drought, salinity), ionizing (IR) and UV radiation and genotoxins, which directly or indirectly via generation of reactive oxidative species (ROS) damage their DNA. Radiomimetic Bleomycin functions as a catalyst to produce ROS leading to clusters of oxidized DNA lesions, single (SSB) and double (DSBs) strand breaks similarly as IR (Steighner & Povirk, 1990). Incurred SSBs and DSBs are easily distinguished and measured by comet assay when varying conditions of mainly used protocol with electrophoresis in 0.3 M NaOH, pH>13 solution (A/A assay). DSBs are detected under “neutral” conditions by N/N assay in regular electrophoretic buffer (Kozak et al, 2009; Olive & Banath, 2006), whilst SSBs are revealed by A/N assay, with alkali-unwinding step in 0.3 M NaOH prior electrophoresis (Angelis et al, 1999; Menke et al, 2001). Better resolution in DSBs and SSBs assays is observed when “neutral” conditions are set between pH 9-10, still well bellow DNA denaturing pH<11.6 (Bradley & Kohn, 1979). Repair of DSBs.DSBs are one of the most cytotoxic forms of DNA damage that must be repaired by recombination, predominantly via non-homologous joining of DNA ends (NHEJ) in higher eukaryotes. However, analysis of DSB repair kinetics of Arabidopsis NHEJ mutants atlig4 and atku80 with the N/N assay showed that repair of Bleomycin induced DSB is biphasic and rapid alternative repair pathways is active (Fig. 1). Surprisingly, kinetic measurements showed that rapid DSB repair was faster in the NHEJ mutant lines (t1/2 5.5 min.) than in wild-type Arabidopsis (t1/2 7.9 min.). Kozak et al. (Kozak et al, 2009) provided the first characterization of this alternate KU/LIG4-independent repair pathway that rapidly removes the majority of DSBs present in nuclear DNA and found its dependence on components of structural maintenance of chromosomes (SMC) complexes, namely SMC6b (MIM) of SMC5/6 complex, kleisin AtRAD21.1 and 3 of cohesin SMC1/3 complex (da Costa-Nunes et al, 2014) and structurally related SMCHD protein GMI1 (Bohmdorfer et al, 2011). Moreno-Romero et al. (Moreno-Romero et al, 2012) studied CK2 protein required for maintenance and control of genomic stability and used N/N assay to show that DSBs were more rapidly repaired in atck2 mutant than in control plants. Their results suggest that atck2 plants are more proficient in repairing DSBs and other lesions produced by IR or Bleomycin, despite their hypersensitivity to these agents.The moss Physcomitrella patens is unique besides the high frequency of homologous recombination for haploid state and filamentous growth during early stages of the vegetative growth. Sheared filaments enables to establish protonemal cultures with up to 50% dividing cells and makes Physcomitrella an excellent model plant to study DNA damage responses. Kamisugi et al. (Kamisugi et al, 2012) used N/N assay to study Physcomitrella mutants of pivotal DSB repair MRN complex (MRE11, RAD50, NBS1). Kinetics of DNA-DSB repair in wild-type and mutant plants revealed that Bleomycin-induced fragmentation of genomic DNA was quickly repaired at approximately equal rates (t1/2 3 min) in each genotype, although both, the ppmre11 and pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and Bleomycin-induced DNA damage. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; it is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development, the similar phenomenon as observed by Moreno-Romero et al. (Moreno-Romero et al, 2012) in atck2 plants. When MRN complex and CK2, both assumed to be associated with error-free homologous recombination are disabled, then NHEJ error-prone repair prevails at a cost of induced mutations in continuous DNA. And indeed this was proved by sequencing of mutated APT locus in which we identified deletions of various lengths as could be expected as an outcome of NHEJ repair mechanism. Combination of comet and mutation assays explained contradictory observation of rapid DSB repair in mutants with phenotype sensitive to induction of DSBs. Evidently plants evolved this attitude of rapid reconstitution of genome integrity from the early evolution stages (mosses were one of the first plants to colonize land) to higher eukaryote seed plants, where during every plant cycle embryos undergo severe DNA damage during seed maturation (generated by ROS released upon desiccation) and efficient recovery during seed germination.Repair of SSBs.