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

Kim, Daria V.; Kulishova, Liliya; Torgasheva, N. A.; Melentyev, Vasily S.; Dianov, Grigory L.; Medvedev, S. P.; Zakian, Suren M.; Zharkov, Dmitry O.

doi:10.1371/journal.pone.0257473

Cited by 6 publications

(16 citation statements)

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“…Interestingly, emerging studies reported that partial Ape1 deficiency did not detectably affect the replication of HeLa cells, CH12F3 cells, HEK293 FT cells, HCT116 cells, and HCC1937 cells [ 57 , 58 , 59 , 60 ]. The first report of a viable APEX1 -KO was in a study addressing the role of Ape1 in AID-mediated class switch recombination in Ig genes [ 61 ], in which a rescue vector expressing Ape1 was maintained while all 3 APEX1 copies in the hypotriploid mouse line CH12F3 were deleted.…”

Section: Ape1 Overviewmentioning

confidence: 99%

“…Surprisingly, removal of the rescue plasmid did not affect cell proliferation, although the complete APEX1 -KO did sensitize cells to the simple alkylating agent methylmethane sulfonate (MMS) [ 61 ]. Recently, a total knockout of APEX1 in (human) HEK293 FT cells (which also have three copies of the gene) gave a very similar result to that of CH12F3 cells; there was no impact on cell proliferation and only a modest increase in MMS sensitivity [ 59 ]. A severe Ape1 knockdown leads to more unrepaired AP sites in genomic DNA [ 56 ], but the mRNA levels of both monofunctional DNA glycosylases (e.g., MutyH, Ung) and multifunctional DNA glycosylases (e.g., Ogg1 and Neil2) were not significantly changed [ 54 , 59 , 62 ].…”

Section: Ape1 Overviewmentioning

confidence: 99%

“…Recently, a total knockout of APEX1 in (human) HEK293 FT cells (which also have three copies of the gene) gave a very similar result to that of CH12F3 cells; there was no impact on cell proliferation and only a modest increase in MMS sensitivity [ 59 ]. A severe Ape1 knockdown leads to more unrepaired AP sites in genomic DNA [ 56 ], but the mRNA levels of both monofunctional DNA glycosylases (e.g., MutyH, Ung) and multifunctional DNA glycosylases (e.g., Ogg1 and Neil2) were not significantly changed [ 54 , 59 , 62 ]. Thus, DNA glycosylase expression was not changed to compensate for the Ape1 deficiency and the extra unrepaired AP sites most likely reflect sluggish BER, with an accumulation of this intermediate.…”

Section: Ape1 Overviewmentioning

confidence: 99%

“…We addressed the question of Ape1 inhibitor targeting by employing the two published APEX1 -KO cell lines—mouse CH12F3 [ 61 ] and human HEK293 FT [ 59 ]—both of which appear to function as well without Ape1 protein as they do with it. We verified the APEX1 -KO lines by Western blotting, which confirmed the absence of Ape1 protein ( Figure 1 A).…”

Section: Evaluation Of Compound 3 and Apx2009 For Possible Off-target...mentioning

confidence: 99%

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Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression

Xue

Demple

2022

Antioxidants

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Apurinic/apyrimidinic endonuclease 1/redox effector-1 (Ape1/Ref-1) is the major apurinic/apyrimidinic (AP) endonuclease in mammalian cells. It functions mainly in the base excision repair pathway to create a suitable substrate for DNA polymerases. Human Ape1 protein can activate some transcription factors to varying degrees, dependent on its N-terminal, unstructured domain, and some of the cysteines within it, apparently via a redox mechanism in some cases. Many cancer studies also suggest that Ape1 has potential for prognosis in terms of the protein level or intracellular localization. While homozygous disruption of the Ape1 structural gene APEX1 in mice causes embryonic lethality, and most studies in cell culture indicate that the expression of Ape1 is essential, some recent studies reported the isolation of viable APEX1 knockout cells with only mild phenotypes. It has not been established by what mechanism the Ape1-null cell lines cope with the endogenous DNA damage that the enzyme normally handles. We review the enzymatic and other activities of Ape1 and the recent studies of the properties of the APEX1 knockout lines. The APEX1 deletions in CH12F3 and HEK293 FT provide an opportunity to test for possible off-target effects of Ape1 inhibition. For this work, we tested the Ape1 endonuclease inhibitor Compound 3 and the redox inhibitor APX2009. Our results confirmed that both APEX1 knockout cell lines are modestly more sensitive to killing by an alkylating agent than their Ape1-proficient cells. Surprisingly, the knockout lines showed equal sensitivity to direct killing by either inhibitor, despite the lack of the target protein. Moreover, the CH12F3 APEX1 knockout was even more sensitive to Compound 3 than its APEX1 + counterpart. Thus, it appears that both Compound 3 and APX2009 have off-target effects. In cases where this issue may be important, it is advisable that more specific endpoints than cell survival be tested for establishing mechanism.

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Section: Ape1 Overviewmentioning

confidence: 99%

Section: Ape1 Overviewmentioning

confidence: 99%

Section: Ape1 Overviewmentioning

confidence: 99%

Section: Evaluation Of Compound 3 and Apx2009 For Possible Off-target...mentioning

confidence: 99%

See 2 more Smart Citations

Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression

Xue

Demple

2022

Antioxidants

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“…Brain-specific knockout mice develop normally in utero but die soon after birth from massive oxidative brain damage [ 34 ]. Several knockout cell lines of human and mouse origin have been reported, their phenotypes ranging from rather mild sensitivity to genotoxic agents to quick, spontaneous apoptosis [ 35 , 36 , 37 , 38 , 39 ].…”

Section: Introductionmentioning

confidence: 99%

Back-Up Base Excision DNA Repair in Human Cells Deficient in the Major AP Endonuclease, APE1

Kim,

Diatlova,

Zharkov

et al. 2023

IJMS

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Apurinic/apyrimidinic (AP) sites are abundant DNA lesions generated both by spontaneous base loss and as intermediates of base excision DNA repair. In human cells, they are normally repaired by an essential AP endonuclease, APE1, encoded by the APEX1 gene. Other enzymes can cleave AP sites by either hydrolysis or β-elimination in vitro, but it is not clear whether they provide the second line of defense in living cells. Here, we studied AP site repairs in APEX1 knockout derivatives of HEK293FT cells using a reporter system based on transcriptional mutagenesis in the enhanced green fluorescent protein gene. Despite an apparent lack of AP site-processing activity in vitro, the cells efficiently repaired the tetrahydrofuran AP site analog resistant to β-elimination. This ability persisted even when the second AP endonuclease homolog, APE2, was also knocked out. Moreover, APEX1 null cells were able to repair uracil, a DNA lesion that is removed via the formation of an AP site. If AP site hydrolysis was chemically blocked, the uracil repair required the presence of NTHL1, an enzyme that catalyzes β-elimination. Our results suggest that human cells possess at least two back-up AP site repair pathways, one of which is NTHL1-dependent.

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Apurinic/apyrimidinic endonuclease 1 has major impact in prevention of suicidal covalent DNA–protein crosslink with apurinic/apyrimidinic site in cellular extracts

Lebedeva,

Dyrkheeva,

Rechkunova

et al. 2024

IUBMB Life

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DNA–protein crosslinks (DPC) are common DNA lesions induced by various external and endogenous agents. One of the sources of DPC is the apurinic/apyrimidinic site (AP site) and proteins interacting with it. Some proteins possessing AP lyase activity form covalent complexes with AP site‐containing DNA without borohydride reduction (suicidal crosslinks). We have shown earlier that tyrosyl‐DNA phosphodiesterase 1 (TDP1) but not AP endonuclease 1 (APE1) is able to remove intact OGG1 from protein–DNA adducts, whereas APE1 is able to prevent the formation of DPC by hydrolyzing the AP site. Here we demonstrate that TDP1 can remove intact PARP2 but not XRCC1 from covalent enzyme–DNA adducts with AP‐DNA formed in the absence of APE1. We also analyzed an impact of APE1 and TDP1 on the efficiency of DPC formation in APE1−/− or TDP1−/− cell extracts. Our data revealed that APE1 depletion leads to increased levels of PARP1–DNA crosslinks, whereas TDP1 deficiency has little effect on DPC formation.

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

Cited by 6 publications

References 93 publications

Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression

Knockout and Inhibition of Ape1: Roles of Ape1 in Base Excision DNA Repair and Modulation of Gene Expression

Back-Up Base Excision DNA Repair in Human Cells Deficient in the Major AP Endonuclease, APE1

Apurinic/apyrimidinic endonuclease 1 has major impact in prevention of suicidal covalent DNA–protein crosslink with apurinic/apyrimidinic site in cellular extracts

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