| Autor: |
Khodyreva SN; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia., Ilina ES; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia.; Faculty of Natural Sciences, Novosibirsk State University, 2 Pirogova Str., Novosibirsk 630090, Russia., Dyrkheeva NS; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia.; Faculty of Natural Sciences, Novosibirsk State University, 2 Pirogova Str., Novosibirsk 630090, Russia., Kochetkova AS; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia., Yamskikh AA; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia.; Faculty of Natural Sciences, Novosibirsk State University, 2 Pirogova Str., Novosibirsk 630090, Russia., Maltseva EA; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia., Malakhova AA; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia.; Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 10 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia., Medvedev SP; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia.; Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 10 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia., Zakian SM; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia.; Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 10 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia., Lavrik OI; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Akad. Lavrentyeva Ave., Novosibirsk 630090, Russia.; Faculty of Natural Sciences, Novosibirsk State University, 2 Pirogova Str., Novosibirsk 630090, Russia. |
| Abstrakt: |
Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1's regulatory functions extend to many cellular processes including the 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. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins-PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase β, DNA ligase III, and XRCC1-did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either. |
