TY - JOUR

T1 - A Knockout of Poly(ADP-Ribose) Polymerase 1 in a Human Cell Line: An Influence on Base Excision Repair Reactions in Cellular Extracts

AU - Khodyreva, Svetlana N.

AU - Ilina, Ekaterina S.

AU - Dyrkheeva, Nadezhda S.

AU - Kochetkova, Alina S.

AU - Yamskikh, Alexandra A.

AU - Maltseva, Ekaterina A.

AU - Malakhova, Anastasia A.

AU - Medvedev, Sergey P.

AU - Zakian, Suren M.

AU - Lavrik, Olga I.

N1 - This study was supported by the Russian Science Foundation (project No. 19-04-00204 for CRISPR/Cas9 editing in cell lines and their characterization and project No. 21-64-00017 for all functional studies) and by the Russian state-funded project for ICBFM SB RAS (grant No. 121031300041-4, for use of the institutional facility for DNA sequencing).

PY - 2024/2

Y1 - 2024/2

N2 - 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.

AB - 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.

KW - CRISPR/Cas9

KW - base excision repair enzymatic activity

KW - mRNA

KW - poly(ADP-ribose) polymerase 1

KW - poly(ADP-ribosyl)ation

UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85185900917&origin=inward&txGid=f7d4ac425102c50bc10f150bb88fa36c

UR - https://www.webofscience.com/wos/woscc/full-record/WOS:001172300000001

UR - https://www.mendeley.com/catalogue/605093c3-d6f4-3dd2-ba7d-35b553194ec2/

U2 - 10.3390/cells13040302

DO - 10.3390/cells13040302

M3 - Article

C2 - 38391916

VL - 13

JO - Cells

JF - Cells

SN - 2073-4409

IS - 4

M1 - 302

ER -