DNA repair in plants studied by comet assay
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1
Institute of Experimental Botany AS CR, Czechia
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2
Institute of Organic Chemistry and Biochemistry AS CR, Czechia
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.). Kozák 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.
Induction of DSB in not an isolated event and depends on inducing agent. Often DSB is a result of clustered damage induced e.g. by impact of radiation beam on DNA or by interaction of agents like Bleomycin with DNA.
Holá et al. (Hola et al, 2013) used N/N and A/N assays to find out whether the repair kinetics of Physcomitrella wt and pplig4 differ in response to Bleomycin-induced DSBs and SSBs. In both lines, the Bleomycin induced DSBs are rapidly repaired with a biphasic kinetic (Fig. 2A) and the half-lives of DSBs survival are similar to half-lives of other Physcomitrella mutants mentioned above.
Contrary to DSBs, SSBs are repaired far less efficiently. Slow SSB repair might be common feature of plants since Donà et al. (Donà et al, 2013) using N/N and A/A assays described similar phenomenon in Medicago truncata cells irradiated with □□-ray. The SSB repair kinetic in wild type Physcomitrella is clearly biphasic and in this respect parallels repair of MMS induced SSBs in wild type Arabidopsis (Waterworth et al, 2009). In contrast to DSBs, substantially smaller fraction of SSBs is repaired with fast kinetics; the defect even more manifested in pplig4. It suggests an important role for LIG4 in the repair of DNA lesions like modified basis, AP sites that are usually detected as SSBs and are repaired via base excision repair (BER). It is noteworthy that LIG3 is not represented in plants and as showed earlier (Waterworth et al, 2009) principal substitute for LIG3 in BER is LIG1. Because LIG1 is essential for cell viability, atlig1 was generated as an RNAi line with 40% of remaining LIG1 activity. SSBs repair course in atlig1 is a consequence of unbalanced BER due to attenuated ligation step leading to accumulation of breaks during early stages of recovery followed by their later gradual removal. Evidently the knockout mutation in pplig4 does not have such severe effect on repair of SSBs; nevertheless, the SSB repair defect of pplig4 clearly manifests that LIG4 is also involved in the repair of SSBs in plants. Effect of break accumulation during early stages recovery is not unique to LIG mutants. Similar effect is depicted on Fig. 2B in Arabidopsis plants with PARP1 impaired either by knockout mutation leading to atparp1 or by two inhibitors, the selective PARP1 inhibitor AG14361 devised by Pfizer for sensitization of cancer cells prior irradiation treatment or unspecific PARP inhibitor 3-aminobenzamide (3-ABA). It is interesting to point out the conservation of PARP system between kingdoms, since selective AG14361 inhibitor of HsPARP1 is also effective in Arabidopsis and provides same repair phenotype as knockout AtPARP1 mutation.
Conclusions
Plant comet assay proved to be a reliable method for studies of damage and the repair of genomic DNA, like in mammals and humans also in plants. Rapid isolation of nuclei from virtually any plant tissue enables to study rapid response of plant cells to DNA damage at virtually any conditions. Comet assay is now successfully applied for basic and applied research from lower (algae, moss) to higher eukaryotic crop plants (rice, wheat). However, even in time of contemporary boom of indirect methods to study repair, direct, unbiased measurement of the integrity of genomic DNA remains beloved domain of the comet assay over counting of γH2AX foci, whose formation depends anyway on phosphorylation signaling cascade.
Fig. 1. Kinetic of DSB repair in Arabidopsis. Fractions of remaining DSBs were calculated for 0, 5, 10, 20 and 60 min repair time points after the treatment with 30 μg/mL Bleomycin. Maximum damage is normalized as 100% at t = 0 for all lines. Wild type Col0 and atlig4 rapidly repair induced DSBs during the first 10 min, while atlig4 has even faster repair rates than wild type. Contrary to wild type, atlig1 and atsmc6b have clearly slower initial DSB repair, with a striking repair defect in SMC6B mutant. Adopted from (Kozak et al, 2009).
Fig. 2A. SSB and DSB repair kinetics in Physcomitrella. SSBs were induced in Physcomitrella protonemata with 50% of dividing cells by 1 hr treatment with 2 □□g/mL Bleomycin; bright blue: wild type, dark blue: pplig4, and determined by A/N assay. DSBs were induced in the same tissue by 1 hr treatment with 30 □□g/mL Bleomycin, green: wild type, orange: pplig4, and determined by N/N assay. Repair kinetics is plotted as % of DSBs remaining over the 0, 5, 10, 20, 60, 180, and 360 min recovery period. Maximum damage is normalized as 100% at □□ = 0 for all lines. Adopted from (Hola et al, 2013).
Fig. 2B. Effect of mutation and of inhibitors of PARP1 on SSB repair kinetics. SSBs induced by 1 hr treatment with 2 mM MMS in atparp1 (red) and in Arabidopsis wt in presence of 3 mM 3-aminobenzamide (3-ABA, turquoise) and 10 μM HsPARP1 specific AG14361 (green) inhibitors. (Angelis and Kozák, unpublished data)
Acknowledgements
Supported by Czech Science Foundation (13-06595S), FP-7-PEOPLE-IRSES-2013 Project # 612587 called: "Plant DNA Tolerance” grants and COST Action CM1201 “Biomimetic Radical Chemistry”. Authors wish to acknowledge indispensable laboratory help of Ms Petra Rožnovská.
References
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Keywords:
physcomitrella patens,
Arabidopsis,
alternative DSB repair,
APT mutation assay,
Smc proteins,
PARP1 mutation and inhibition,
DSB detection,
SSB detection
Conference:
ICAW 2015 - 11th International Comet Assay Workshop, Antwerpen, Belgium, 1 Sep - 4 Sep, 2015.
Presentation Type:
Oral Presentation
Topic:
Ecogenotoxicology
Citation:
Angelis
KJ,
Kozák
J,
Vágnerová
R and
Holá
M
(2015). DNA repair in plants studied by comet assay.
Front. Genet.
Conference Abstract:
ICAW 2015 - 11th International Comet Assay Workshop.
doi: 10.3389/conf.fgene.2015.01.00067
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Received:
06 May 2015;
Published Online:
23 Jun 2015.
*
Correspondence:
Dr. Karel J Angelis, Institute of Experimental Botany AS CR, Praha, Czechia, karel.angelis@gmail.com