DNA repair in plants studied by comet assay

0447959 - ÚEB 2016 CH eng C - Conference Paper (international conference)
Angelis, Karel - Kozák, Jaroslav - Vágnerová, Radka - Holá, Marcela
DNA repair in plants studied by comet assay.
Frontiers in Genetics Conference. Lausanne: ICAW, 2015 - (Allison, D.). E-ISSN 1664-8021.
[International Comet Assay Workshop /11./. Antwerpen (BE), 01.09.2015-04.09.2015]
R&D Projects: GA ČR GA13-06595S
Institutional support: RVO:61389030 ; RVO:61388963
Keywords : physcomitrella patens * Arabidopsis * alternative DSB repair
Subject RIV: EB - Genetics ; Molecular Biology

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).
Permanent Link: http://hdl.handle.net/11104/0249688