The Use of qPCR to Evaluate the Efficiency of Bulky DNA Damage Removal in Extracts of Mammalian Cells with Different Maximum Lifespan

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Abstract

Proteins of the nucleotide excision repair (NER) system are responsible for the detection and removal of a wide range of bulky damages from DNA, thereby making a significant contribution to maintaining genome stability in mammalian cells. Diagnosis of the functional status of NER in cells is important for detecting pathological changes in the body and evaluating the efficiency of the use of chemotherapeutic drugs. The paper describes a method for evaluating the efficiency of in vitro removal of bulky DNA damage based on the use of qPCR. Using the developed method, a comparative assessment of NER activity on cell extracts of two mammals with different lifespans – a long-lived naked mole rat (Heterocephalus glaber) and a short-lived mouse (Mus musculus) was carried out. It has been shown that the proteins of the H. glaber cell extract are 1.5 times more effective at removing bulky damage from the model DNA substrate than the proteins of the M. musculus cell extract, which is consistent with the experimental data obtained earlier. The presented development can be used not only in fundamental research of DNA repair in mammalian cells, but also in clinical practice.

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About the authors

A. A. Popov

Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences

Email: lavrik@niboch.nsc.ru
Russian Federation, Novosibirsk

V. A. Shamanin

BioLink LLC

Email: lavrik@niboch.nsc.ru
Russian Federation, Novosibirsk

I. O. Petruseva

Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences

Email: lavrik@niboch.nsc.ru
Russian Federation, Novosibirsk

A. N. Evdokimov

Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences

Email: lavrik@niboch.nsc.ru
Russian Federation, Novosibirsk

O. I. Lavrik

Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences; Novosibirsk National Research State University

Author for correspondence.
Email: lavrik@niboch.nsc.ru
Russian Federation, Novosibirsk; Novosibirsk

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Supplementary files

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2. Fig. 1. Chemical structures of nFlu (a) and TEG (b)

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3. Fig. 2. Schematic representation of the proposed approach to assess the efficiency of in vitro bulk lesion removal by qPCR

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4. Fig. 3. Analysis of nFlu/TEG- and nm/TEG-DNA amplification results. Amplification curves of nm/TEG-DNA standard samples (a), calibration graph (b) and results of nm/TEG-DNA amplification efficiency calculation (c) are presented. d - Example of nm/TEG- and nFlu/TEG-DNA amplification curves; e - mean values of C(t) and standard deviation for nm/TEG- and nFlu/TEG-DNA obtained from three measurements; f - melting curves of nm/TEG- and nFlu/TEG-DNA amplification products

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5. Fig. 4. Comparative evaluation of nFlu/TEG-DNA repair efficiency by proteins of Heterocephalus glaber and Mus musculus cell extracts using qPCR. Amplification curves of nm/TEG-DNA (purple), nFlu/TEG-DNA (green), and nFlu/TEG-DNA after 30 min incubation with extract proteins (dark green) obtained for H. glaber (a) and M. musculus (b); c, comparison of nFlu/TEG-DNA repair efficiency by NER proteins of cell extract from H. glaber (a) and M. musculus (b). glaber (blue) and M. musculus (red) as a function of incubation time; d - diagram showing the differences in the efficiency of nFlu/TEG-DNA repair by NER proteins of H. glaber cell extract (blue) and M. musculus (red). glaber (blue) and M. musculus (red) after 30 min of incubation; e - evaluation of the effect of H. glaber and M. musculus cell extract proteins on nm/TEG-DNA repair. musculus on nm/TEG-DNA free of bulk damage after 30 min of incubation; f - example of melting curves of amplification products of nm/TEG-DNA (purple), nFlu/TEG-DNA (green) and nFlu/TEG-DNA after 30 min of incubation with NER proteins (dark green) of M. musculus cell extract. Results of three biological repeats are presented with standard deviation, *** p < 0.001

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