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Response of PRIMPOL-Knockout Human Lung Adenocarcinoma A549 Cells to Genotoxic Stress

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Abstract

Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.

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Abbreviations

EdU:

5-ethynyl-2′-deoxyuridine

MMS:

methyl methanesulfonate

MTT:

methyl tetrazolium

PI:

propidium iodide

PrimPol:

primase-polymerase

RAD51:

RAD51 recombinase

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Funding

This work was supported by the Russian Science Foundation (grant no. 18-14-00354 for A.V.M.; generation of PRIMPOL–/– cells, analysis of metabolic activity and DNA-replicating cell fractions, cell cycle analysis) and the Russian Foundation for Basic Research (grant no. 20-34-90092-Aspirants for D.V.K.; generation of clonal cell lines), and by the Ministry of Science and Higher Education of the Russian Federation (State Assignment 075-03-2023-106, project no. FSMG-2023-0015 for M.V.P.; cell treatment with ionizing radiation).

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    Authors and Affiliations

    1. Institute of Molecular Genetics, Kurchatov Institute National Research Center, 123182, Moscow, Russia

      Anastasia S. Gromova, Elizaveta O. Boldinova & Alena V. Makarova

    2. Institute of Gene Biology, Russian Academy of Sciences, 119334, Moscow, Russia

      Anastasia S. Gromova, Elizaveta O. Boldinova & Alena V. Makarova

    3. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090, Novosibirsk, Russia

      Daria V. Kim & Dmitry O. Zharkov

    4. Novosibirsk State University, 630090, Novosibirsk, Russia

      Daria V. Kim & Dmitry O. Zharkov

    5. School of Biological and Medical Physics, Moscow Institute of Physics and Technology, 141700, Dolgoprudny, Russia

      Roman N. Chuprov-Netochin, Sergey V. Leonov & Margarita V. Pustovalova

    6. Institute of Cell Biophysics of the Russian Academy of Sciences, 142290, Pushchino, Russia

      Sergey V. Leonov

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    1. Anastasia S. Gromova
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    Contributions

    A.S.G. performing experiments, data analysis, writing the draft; E.O.B. performing experiments, data analysis, writing the draft; D.V.K. planning and performing experiments, funding; R.N.C. planning and performing experiments, S.V.L. correcting the article and funding, M.V.P. planning and performing experiments, data analysis, writing the draft; D.O.Z. planning experiments and correcting the article, A.V.M. planning experiments, data analysis, writing the draft and correcting the article, funding.

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    Correspondence to Dmitry O. Zharkov or Alena V. Makarova.

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    The authors declare no conflict of interest. This article does not contain description of research involving humans or animals as subjects performed by any of the authors.

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    Gromova, A.S., Boldinova, E.O., Kim, D.V. et al. Response of PRIMPOL-Knockout Human Lung Adenocarcinoma A549 Cells to Genotoxic Stress. Biochemistry Moscow 88, 1933–1943 (2023). https://doi.org/10.1134/S0006297923110214

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