Method for assessment of nucleotide excision repair system efficiency ex vivo

Abstract

The nucleotide excision repair (NER) is one of the main repair systems present in the cells of living organisms. It is responsible for the removal of a wide range of bulky DNA lesions. We succeeded in developing a method for assessing the efficiency of NER in the cell (ex vivo), which is a method based on the recovery of TagRFP fluorescent protein production through repair of the damage that blocks the expression of the appropriate gene. Our constructed plasmids containing bulky nFlu or nAnt lesions near the tagrfp gene promoter were shown to undergo repair in eukaryotic cells (HEK 293T) and that they can be used to analyze the efficiency of NER ex vivo. A comparative analysis of the time dependence of fluorescent cells accumulation after transfection with nFlu- and nAnt-DNA revealed that there are differences in how efficient their repair by the NER system of HEK 293T cells can be. The method can be used to assess the cell repair status and the repair efficiency of different structural damages.