Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Kim, Daria V., Kulishova, Liliya M., Torgasheva, Natalia A., Melentyev, Vasily S., Dianov, Grigory L., Medvedev, Sergey P., Zakian, Suren M., Zharkov, Dmitry O.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8445474/
https://www.ncbi.nlm.nih.gov/pubmed/34529719
http://dx.doi.org/10.1371/journal.pone.0257473
_version_ 1784568667765211136
author Kim, Daria V.
Kulishova, Liliya M.
Torgasheva, Natalia A.
Melentyev, Vasily S.
Dianov, Grigory L.
Medvedev, Sergey P.
Zakian, Suren M.
Zharkov, Dmitry O.
author_facet Kim, Daria V.
Kulishova, Liliya M.
Torgasheva, Natalia A.
Melentyev, Vasily S.
Dianov, Grigory L.
Medvedev, Sergey P.
Zakian, Suren M.
Zharkov, Dmitry O.
author_sort Kim, Daria V.
collection PubMed
description 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 EC(50) 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.
format Online
Article
Text
id pubmed-8445474
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-84454742021-09-17 Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line Kim, Daria V. Kulishova, Liliya M. Torgasheva, Natalia A. Melentyev, Vasily S. Dianov, Grigory L. Medvedev, Sergey P. Zakian, Suren M. Zharkov, Dmitry O. PLoS One Research Article 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 EC(50) 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. Public Library of Science 2021-09-16 /pmc/articles/PMC8445474/ /pubmed/34529719 http://dx.doi.org/10.1371/journal.pone.0257473 Text en © 2021 Kim et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Kim, Daria V.
Kulishova, Liliya M.
Torgasheva, Natalia A.
Melentyev, Vasily S.
Dianov, Grigory L.
Medvedev, Sergey P.
Zakian, Suren M.
Zharkov, Dmitry O.
Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line
title Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line
title_full Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line
title_fullStr Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line
title_full_unstemmed Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line
title_short Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line
title_sort mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease apex1 in a non-cancer human cell line
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8445474/
https://www.ncbi.nlm.nih.gov/pubmed/34529719
http://dx.doi.org/10.1371/journal.pone.0257473
work_keys_str_mv AT kimdariav mildphenotypeofknockoutsofthemajorapurinicapyrimidinicendonucleaseapex1inanoncancerhumancellline
AT kulishovaliliyam mildphenotypeofknockoutsofthemajorapurinicapyrimidinicendonucleaseapex1inanoncancerhumancellline
AT torgashevanataliaa mildphenotypeofknockoutsofthemajorapurinicapyrimidinicendonucleaseapex1inanoncancerhumancellline
AT melentyevvasilys mildphenotypeofknockoutsofthemajorapurinicapyrimidinicendonucleaseapex1inanoncancerhumancellline
AT dianovgrigoryl mildphenotypeofknockoutsofthemajorapurinicapyrimidinicendonucleaseapex1inanoncancerhumancellline
AT medvedevsergeyp mildphenotypeofknockoutsofthemajorapurinicapyrimidinicendonucleaseapex1inanoncancerhumancellline
AT zakiansurenm mildphenotypeofknockoutsofthemajorapurinicapyrimidinicendonucleaseapex1inanoncancerhumancellline
AT zharkovdmitryo mildphenotypeofknockoutsofthemajorapurinicapyrimidinicendonucleaseapex1inanoncancerhumancellline