Response of PRIMPOL-Knockout Human Lung Adenocarcinoma A549 Cells to Genotoxic Stress.

Human DNA primase/polymerase PrimPol synthesizes DNA primers de novo after replication fork stalling at the sites of DNA damage, thus contributing to the DNA damage tolerance. The role of PrimPol in response to the different types of DNA damage is poorly understood. We knocked out the PRIMPOL gene in the lung carcinoma A549 cell line and characterized the response of the obtained cells to the DNA damage caused by hydrogen peroxide, methyl methanesulfonate (MMS), cisplatin, bleomycin, and ionizing radiation. The PRIMPOL knockout reduced the number of proliferating cells and cells in the G2 phase after treatment with MMS and caused a more pronounced delay of the S phase in the cisplatin-treated cells. Ionizing radiation at a dose of 10 Gy significantly increased the content of apoptotic cells among the PRIMPOL-deficient cells, while the proportion of cells undergoing necroptosis increased in both parental and knockout cells at any radiation dose. The viability of PRIMPOL-deficient cells upon the hydrogen peroxide-induced oxidative stress increased compared to the control cells, as determined by the methyl tetrazolium (MTT) assay. The obtained data indicate the involvement of PRIMPOL in the modulation of adaptive cell response to various types of genotoxic stress.

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair.

CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

Abasic site-peptide cross-links are blocking lesions repaired by AP endonucleases.

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA-protein cross-links. Those are subject to proteolysis but the fate of the resulting AP-peptide cross-links (APPXLs) is unclear. Here, we report two in vitro models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, Saccharolobus solfataricus Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, Escherichia coli endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, E. coli exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.

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

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 ß-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 ß-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 ß-elimination. Our results suggest that human cells possess at least two back-up AP site repair pathways, one of which is NTHL1-dependent.

Recognition of a Clickable Abasic Site Analog by DNA Polymerases and DNA Repair Enzymes.

Azide-alkyne cycloaddition ("click chemistry") has found wide use in the analysis of molecular interactions in living cells. 5-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (EAP) is a recently developed apurinic/apyrimidinic (AP) site analog functionalized with an ethynyl moiety, which can be introduced into cells in DNA constructs to perform labeling or cross-linking in situ. However, as a non-natural nucleoside, EAP could be subject to removal by DNA repair and misreading by DNA polymerases. Here, we investigate the interaction of this clickable AP site analog with DNA polymerases and base excision repair enzymes. Similarly to the natural AP site, EAP was non-instructive and followed the "A-rule", directing residual but easily detectable incorporation of dAMP by E. coli DNA polymerase I Klenow fragment, bacteriophage RB69 DNA polymerase and human DNA polymerase ß. On the contrary, EAP was blocking for DNA polymerases κ and λ. EAP was an excellent substrate for the major human AP endonuclease APEX1 and E. coli AP exonucleases Xth and Nfo but was resistant to the AP lyase activity of DNA glycosylases. Overall, our data indicate that EAP, once within a cell, would represent a replication block and would be removed through an AP endonuclease-initiated long-patch base excision repair pathway.

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 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.

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

Base excision DNA repair (BER) is a vitally important pathway that protects the cell genome from many kinds of DNA damage, including oxidation, deamination, and hydrolysis. It involves several tightly coordinated steps, starting from damaged base excision and followed by nicking one DNA strand, incorporating an undamaged nucleotide, and DNA ligation. Deficiencies in BER are often embryonic lethal or cause morbid diseases such as cancer, neurodegeneration, or severe immune pathologies. Starting from the early 1980s, when the first mammalian cell lines lacking BER were produced by spontaneous mutagenesis, such lines have become a treasure trove of valuable information about the mechanisms of BER, often revealing unexpected connections with other cellular processes, such as antibody maturation or epigenetic demethylation. In addition, these cell lines have found an increasing use in genotoxicity testing, where they provide increased sensitivity and representativity to cell-based assay panels. In this review, we outline current knowledge about BER-deficient cell lines and their use.