Abstract
The base excision repair (BER) system is aimed at repair of the largest group of DNA lesions, namely of damaged bases. The main steps in BER are: recognition and removal of the aberrant base, cutting the DNA sugar-phosphate backbone, gap processing (including dNMP insertion), and DNA ligation. The precise functioning of BER depends on the regulation of each step of the process by regulatory/accessory proteins, the most important of which is poly(ADP-ribose)polymerase 1 (PARP1). PARP1 plays an important role in various processes of DNA repair, maintenance of genome integrity, and regulation of mRNA stability and decay. In this regard, PARP1 can affect BER both at the level of proteins involved in the process and at the level of expression of the mRNAs encoding them. There are no systematic data on the effect of various amounts of PARP1 on the activity of key BER proteins and the levels of mRNAs encoding them in human cells. In our work, using whole-cell extracts and RNA preparations obtained from the parental HEK293T cell line and its derivative HEK293T/P1-KD cell line with reduced PARP1 expression (shPARP1-expressing cells, PARP1 knockdown), we assessed the levels of mRNA encoding BER proteins: PARP1, PARP2, uracil DNA glycosylase (UNG2), AP endonuclease 1 (APE1), DNA polymerase β (POLβ), DNA ligase III (LIG3), and XRCC1. In parallel, the catalytic activity of these enzymes was evaluated. No significant effect of the PARP1 amount of on the mRNA levels of UNG2, APE1, POLβ, LIG3, and XRCC1 was found. At the same time, in HEK293T/P1-KD cells, the amount of PARP2 mRNA was reduced by 2 times. The activities of these enzymes in whole-cell extracts of HEK293T and HEK293T/P1-KD cells also did not differ significantly. Under the conditions of poly(ADP-ribose) synthesis, the efficiency of the reactions catalyzed by UNG2, APE1, POLβ, and LIG3 also did not change significantly. In addition, it was shown that a reduced amount of PARP1 in the extract of HEK293T/P1-KD cells does not cause fundamental changes in the nature of DNA PARylation compared to the extract of the parental HEK293T cell line.
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