Single-cell gel/comet assay applied to the analysis of UV radiation-induced DNA damage in Rhodomonas sp (Cryptophyta)

The single-cell gel/comet assay is an electrophoretic technique used to det ect single-strand breaks in DNA, Damage is assessed examining individual ce lls under an epifluorescent microscope. UV-induced DNA damage consists most ly of the formation of pyrimidine dimers; therefore, most of the damage can not be detected using a standard comet assay. The enzyme T4 endonuclease V breaks DNA strands at sites of pyrimidine dimers, The main objective of thi s work is to evaluate the comet assay to detect UV-induced damage in DNA af ter an initial treatment of cells with T4 endonuclease V, This work was con ducted on Rhodomonas sp, (Cryptophyta), a marine unicellular flagellate, Ce lls of Rhodomonas sp, were exposed to 12 h visible + ultraviolet-A + ultrav iolet-B (VIS + UVA + UVB) and VIS (control), with and without T4 endonuclea se V. Cells exposed to VIS + UVA + UVB showed approximately 200% more damag e than control if these were treated with T4 endonuclease V, Rhodomonas sp, were exposed to 3, 6, 9 and 12 h of VIS, VIS + UVA and VIS + UVA + UVB, Da mage induced by VIS + UVA + UVB as detected by the comet assay increased al ong with exposure time. However, damage caused by VIS and VIS + UVA remaine d relatively constant at all times. Results of this study indicate that the comet assay is more sensitive to UV radiation damage when used in conjunct ion with T4 endonuclease V. This modification of the comet assay can be use d as an alternative technique to detect DNA damage in single cells caused b y UV radiation.