Influence of the Poly(ADP-Ribose) Polymerase 1 Level on the Status of Base Excision Repair in Human Cells

Abstract

Base excision repair (BER) is aimed at repair of damaged bases, which are the largest group of DNA lesions. The main steps of BER are recognition and removal of the aberrant base, cutting of the DNA sugar-phosphate backbone, gap processing (including dNMP insertion), and DNA ligation. The precise function of BER depends on the regulation of each step by regulatory/accessory proteins, the most important of which is poly(ADP-ribose) (PAR) polymerase 1 (PARP1). PARP1 plays an important role in DNA repair, maintenance of genome integrity, and regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. There is no systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells. 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, a PARP1 knockdown) were used to assess the levels of mRNAs coding for BER proteins: PARP1, PARP2, uracil DNA glycosylase (UNG2), AP endonuclease 1 (APE1), DNA polymerase β (POLβ), DNA ligase III (LIG3), and XRCC1. Catalytic activities of the enzymes were evaluated in parallel. No significant effect of the PARP1 content was observed for the mRNA levels of UNG2, APE1, POLβ, LIG3, and XRCC1. The amount of the PARP2 mRNA proved to be reduced two times in HEK293T/P1-KD cells. Activities of these enzymes in whole-cell extracts did not differ significantly between HEK293T and HEK293T/P1-KD cells. No significant change was observed in the efficiencies of the reactions catalyzed by UNG2, APE1, POLβ, and LIG3 in conditions of PAR synthesis. A DNA PARylation pattern did not dramatically change in a HEK293T/P1-KD cell extract with a reduced PARP1 content as compared with an extract of the parental HEK293T cell line.