Base Excision DNA Repair Deficient Cells: From Disease Models to Genotoxicity Sensors

Kim, Daria V.; Makarova, Аlena V.; Miftakhova, Regina; Zharkov, Dmitry O.

doi:10.2174/1381612825666190319112930

Cited by 7 publications

(4 citation statements)

References 287 publications

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“…As a result, efficient BER plays a vital role in maintaining genomic integrity. On the other hand, BER failure is both cytotoxic and potentially mutagenic, thus contributing to the pathology of disease 17,[31][32][33] . Akin to genomic DNA, dNTPs are also vulnerable to oxidation by ROS, leading to the presence of their oxidized forms (e.g.…”

Section: Discussionmentioning

confidence: 99%

Processing of oxidatively damaged DNA dirty ends by APE1

Whitaker

Stark

Freudenthal

2021

Preprint

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Reactive oxygen species attack the structure of DNA, thus altering its base-pairing properties. Consequently, oxidative stress-associated DNA lesions are a major source of the mutation load that gives rise to cancer and other diseases. Base excision repair (BER) is the pathway primarily tasked with repairing DNA base damage, with apurinic/apyrimidinic endonuclease (APE1) having both AP-endonuclease and 3’ to 5’ exonuclease (exo) DNA cleavage functions. The lesion 8-oxo-7,8-dihydroguanine (8-oxoG) can enter the genome as either a product of direct damage to the DNA, or through polymerase insertion at the 3’-end of a DNA strand during replication or repair. Importantly, 3’-8-oxoG impairs the ligation step of BER and therefore must be removed by the exo activity of a surrogate enzyme to prevent double stranded breaks and cell death. In the present study, we characterize the exo activity of APE1 on 3’-8-oxoG substrates. These structures demonstrate that APE1 uses a unified mechanism for its exo activities that differs from its more canonical AP-endonuclease activity. In addition, through complementation of the structural data with enzyme kinetics and binding studies employing both wild-type and rationally designed APE1 mutants, we were able to identify and characterize unique protein:DNA contacts that specifically mediate 8-oxoG removal by APE1.

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Section: Discussionmentioning

confidence: 99%

Processing of oxidatively damaged DNA dirty ends by APE1

Whitaker

Stark

Freudenthal

2021

Preprint

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“…Presently, there are few mammalian cell models with stably inactivated genes encoding APEX1 homologs [ 32 ]. A knockout was generated by a conventional recombination approach in the hypotriploid mouse B-cell line CH12F3, producing cells hypersensitive to MMS [ 33 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2021

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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.

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“…Several variations on this general mechanism operate in mammalian cells, differing in the requirements for DNA glycosylases and AP endonucleases and generating single-strand breaks with different termini [2][3][4][5]. Deficiencies in the key components of BER are embryonic lethal [6][7] while certain functional variants are associated with an increased risk of cancer [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Reading Targeted DNA Damage in the Active Demethylation Pathway: Role of Accessory Domains of Eukaryotic AP Endonucleases and Thymine-DNA Glycosylases

Popov

Grin

Dvornikova

et al. 2020

Journal of Molecular Biology

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Base excision DNA repair (BER) is an important process used by all living organisms to remove non-bulky lesions from DNA. BER is usually initiated by DNA glycosylases that excise a damaged base leaving an apurinic/apyrimidinic (AP) site, an AP endonuclease then cuts DNA at the AP site, and the repair is completed by correct nucleotide insertion, end processing, and nick ligation. It has emerged recently that the BER machinery, in addition to genome protection, is crucial for active epigenetic demethylation in vertebrates. This pathway is initiated by TET dioxygenases that oxidize the regulatory 5-methylcytosine, and the oxidation products are treated as substrates for BER. T:G mismatch-specific thymine-DNA glycosylase (TDG) and AP endonuclease 1 (APE1) catalyze the first two steps in BER-dependent active demethylation. In addition to the well-structured catalytic domains, these enzymes possess long tails that are structurally uncharacterized but involved in multiple interactions and regulatory functions. In this review, we describe known roles of the tails in TDG and APE1, discuss the importance of order and disorder in their structure and consider the evolutionary aspects of these accessory protein regions. We also propose that the tails may be important for the enzymes' oligomerization on DNA, an aspect of their function that only recently gained attention.

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Base Excision DNA Repair Deficient Cells: From Disease Models to Genotoxicity Sensors

Cited by 7 publications

References 287 publications

Processing of oxidatively damaged DNA dirty ends by APE1

Processing of oxidatively damaged DNA dirty ends by APE1

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

Reading Targeted DNA Damage in the Active Demethylation Pathway: Role of Accessory Domains of Eukaryotic AP Endonucleases and Thymine-DNA Glycosylases

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