Repair of X-ray induced DNA damage measured by the comet assay in roots of Vicia faba

Abstract

The comet assay was used to measure DNA damage and repair in nuclei released from 1 cm root ends of Vicia faba after X-ray irradiation. Irradiation induced a linear increase of DNA content in comet tail with doses under various denaturation and electrophoretic conditions. The pH of the electrophoresis solution played the most important role in the detection of DNA damage. After irradiation with 30 Gy of X-rays, most of the DNA damage was removed during the first 20 min, even in the presence of DNA repair inhibitors. This first, rapid phase of DNA repair was not affected by incubation on ice, but was partially blocked by 3-aminobenzamide. When DNA was exposed to alkali (0.3 M NaOH) and electrophoresed at neutral pH, all DNA damage was removed in 2 hr, even in the presence of 3-aminobenzamide. Complete repair was inhibited by incubation on ice (30% of DNA remaining in tail) and partially by aphidicolin (13% DNA remaining in tail). Under alkaline (0.3 M NaOH) pretreatment and electrophoresis, more than 20% of detected DNA damage remained unrepaired after 2 hr of postirradiation incubation with and without 3-aminobenzamide at room temperature. Aphidicolin and incubation on ice inhibited the removal of DNA damage to 33% and 39% DNA, respectively. Moreover, aphidicolin treatment attenuated the first phase of damage removal.