Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line

The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell l...

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Published in:	PloS one Vol. 16; no. 9; p. e0257473
Main Authors:	Kim, Daria V, Kulishova, Liliya M, Torgasheva, Natalia A, Melentyev, Vasily S, Dianov, Grigory L, Medvedev, Sergey P, Zakian, Suren M, Zharkov, Dmitry O
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 16-09-2021 Public Library of Science (PLoS)
Subjects:	Aldehydes Alleles Analysis Biology and life sciences Cancer Cell Cycle Checkpoints Cellular biology CRISPR CRISPR-Cas Systems - genetics Cytotoxicity Deoxyribonucleic acid DNA DNA damage DNA polymerase DNA repair DNA Repair - drug effects DNA-(Apurinic or Apyrimidinic Site) Lyase - chemistry DNA-(Apurinic or Apyrimidinic Site) Lyase - genetics DNA-(Apurinic or Apyrimidinic Site) Lyase - metabolism E coli Earth Sciences Embryos Endonuclease Frameshift mutation Gene deletion Gene Editing Gene expression Genetic aspects Genetic transcription HEK293 Cells Human cell culture Humans Hydrogen Hydrogen peroxide Hydrogen Peroxide - chemistry Insertion Laboratories Mammalian cells Mammals Medicine Methyl methanesulfonate Methyl Methanesulfonate - pharmacology Mutation Null cells Oxidation Oxidative stress Oxidizing agents Phenotype Phenotypes Physical Sciences Plasmids Potassium Potassium bromate Proteins Radiation Repair Research and analysis methods RNA, Guide, CRISPR-Cas Systems - metabolism Sensitivity Substrates Toxicity Transcription factors Translation termination Uracil Vertebrates Russia United Kingdom > UK
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Summary:	The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out the APEX1 gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein's catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of the APEX1 transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50 compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5-2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.
Bibliography:	Competing Interests: The authors have declared that no competing interests exist.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0257473