Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Knockout of Poly(ADP-ribose) polymerase 1 in human cell line: Influence on the Base Excision Repair Reactions in the Cellular Extracts

Svetlana N. Khodyreva
* ,
Ekaterina S. Ilina
ORCID logo ,
Nadezhda S. Dyrkheeva
ORCID logo ,
Alina S. Kochetkova
,
Alexandra A. Yamskikh
,
Ekaterina A. Maltseva
,
Anastasia A. Malakhova
ORCID logo ,
Sergey P. Medvedev
ORCID logo ,
Suren M. Zakian
,
Olga I. Lavrik
* ORCID logo
Version 1 : Received: 8 December 2023 / Approved: 12 December 2023 / Online: 12 December 2023 (11:08:39 CET)

A peer-reviewed article of this Preprint also exists.

Khodyreva, S.N.; Ilina, E.S.; Dyrkheeva, N.S.; Kochetkova, A.S.; Yamskikh, A.A.; Maltseva, E.A.; Malakhova, A.A.; Medvedev, S.P.; Zakian, S.M.; Lavrik, O.I. A Knockout of Poly(ADP-Ribose) Polymerase 1 in a Human Cell Line: An Influence on Base Excision Repair Reactions in Cellular Extracts. Cells 2024, 13, 302. Khodyreva, S.N.; Ilina, E.S.; Dyrkheeva, N.S.; Kochetkova, A.S.; Yamskikh, A.A.; Maltseva, E.A.; Malakhova, A.A.; Medvedev, S.P.; Zakian, S.M.; Lavrik, O.I. A Knockout of Poly(ADP-Ribose) Polymerase 1 in a Human Cell Line: An Influence on Base Excision Repair Reactions in Cellular Extracts. Cells 2024, 13, 302.

Abstract

Base excision repair (BER) is the predominant pathway to remove most forms of hydrolytic, oxidative and alkylative DNA damage. The precise functioning of BER is achieving by the regulation of each step by regulatory/accessory proteins, with the most important of which being poly(ADP-ribose) polymerase 1 (PARP1). The PARP1 regulatory functions extend to many cellular processes including regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of the 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 the CRISPR/Cas9 based technique has been used to knocked out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with putative PARP1 deletion were characterized by several approaches including: PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, qPCR analysis of PARP1 mRNA, western-blot analysis of whole-cell-extract proteins with anti-PARP1 antibodies, and the PAR synthesis in the whole-cell-extracts. qPCR analysis of mRNAs coding the BER related proteins: PARP2, uracil DNA glycosylase 2, AP endonuclease 1, DNA polymerase β, DNA ligase III and XRCC1 did not reveal crucial influence of PARP1 knockout. The corresponding catalytic activities in the whole-cell extracts (WCEs) evaluated in parallel did not differ significantly between mutant and parental cell lines. No considerable influence of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed.

Keywords

CRISPR/Cas9 technique; mRNAs; poly(ADP-ribose) polymerase 1; poly(ADP- ribosyl)ation.; activity of base excision repair enzymes

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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