Accepted_test

Nucleotide excision repair (NER) proteins remove a wide range of bulky damages from DNA, including adducts formed by UV light and harmful polycyclic compounds from the environment. The assessment of the functional status of NER in cells is important both for fundamental research and for the diagnosis of various pathological states. In this work a method for evaluating the efficiency of bulky damage removal using qPCR and NER-competent extracts was described. For this purpose, model DNA containing bulky nFlu or nAnt damages in one of the chains and a non-bulk TEG insert in the complementary chain were synthesized. An analysis of the amplification of the obtained DNA substrates using qPCR was performed, and conditions for conducting the NER reaction were selected for the new technique. Using the developed approach, a comparative assessment of the efficiency of removing bulky nFlu damage from model DNA by proteins from cell extracts of a long-lived naked mole rat and a short-lived mouse was carried out. In addition, the efficiency of removing bulky damages of nFlu and nAnt from the corresponding DNA substrates by proteins of the CHO cell extract was evaluated. The results obtained demonstrate the applicability of the developed approach to evaluate the efficiency of removal of bulky DNA damages in vitro.