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

Daria V Kim,Suren M Zakian,Dmitry O Zharkov,Natalia A Torgasheva,Vasily S Melentyev,Liliya M Kulishova,Sergey P Medvedev,Grigory L Dianov,Robert W Sobol

doi:10.1371/journal.pone.0257473

Abstract

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.

Highlights

Many endogenous and environmental factors generate a steady stream of lesions in cellular DNA
Two of the protospacers were located in exon 2 spanning the initiator ATG codon and were shifted by a single base relative to each other, leading to the use of different protospacer-adjacent motif (PAM) sequences: TGG for Protospacer 1 and GGG for Protospacer 2
While many bacteria have two AP endonucleases of unrelated structure but with overlapping enzyme activities (Xth and Nfo in E. coli), APEX1 seems to be the major human AP endonuclease supporting most of Base excision repair (BER) and carrying out additional functions such as stimulation of transcription factors binding to DNA, RNA

Summary

Introduction

Many endogenous and environmental factors generate a steady stream of lesions in cellular DNA. Estimates based on known rates of spontaneous DNA damage suggest that ~20,000 endogenous lesions appear in each cell’s DNA per day [1]. Base excision repair (BER) is the system primarily responsible for the removal of small non-bulky lesions from DNA [2, 3]. BER is initiated by various DNA glycosylases, each of which is specific to a characteristic type of damage. DNA glycosylases recognize damaged bases and hydrolyze their N-glycosidic bonds, leaving a baseless deoxyribose residue (apurinic/apyrimidinic site, or AP site). DNA polymerase β (POLβ) eliminates the blocking 20deoxyribose-50-phosphate and inserts an undamaged dNMP. The complex of XRCC1 adaptor protein and DNA ligase IIIα seals the remaining nick

Methods

Results

Discussion

Conclusion