Enhancing APE1 detection through apurinic/apyrimidinic site inhibition of DNA polymerase: an innovative, highly sensitive approach.

This study presents an innovative method for the highly sensitive detection of apurinic/apyrimidinic endonuclease 1 (APE1), a crucial biomarker and target for cancer diagnosis and treatment. The method is predicated on our discovery that the apurinic or apyrimidinic site (AP site) can inhibit the activity of Taq DNA polymerase. Subsequent experiments further led to the development of a new amplification method based on the digestion activity of Lambda exonuclease. This approach showed potential to detect trace amounts of APE1 in biological samples with high sensitivity.

An enzymatically activated AND-gate DNA logic circuit for tumor cells recognition via multi-microRNAs detection.

The DNA-based logic circuit, constructed to mimic biochemical reaction networks, is highly significant in detecting biomarkers at the molecular level. The differences in the expression levels of microRNAs (miRNAs) within different types of cells provide hope for distinguishing cell subtypes. However, reliance on a single miRNA often leads to unreliable results. Herein, we constructed an enzyme-triggered cascade logic circuit based on the AND gate, which is capable of generating corresponding fluorescence signals in the presence of target miRNAs. The introduction of apurinic/apyrimidinic (AP) sites effectively reduces the likelihood of false signal generation. Amplification of the fluorescence signal relies on the catalytic hairpin assembly and the repetitive reuse of the multicomponent nucleic acid enzyme (MNAzyme). We demonstrated that the logic circuit can not only distinguish cancer cells from normal cells but also identify different types of cancer cells. The programmability of the logic circuits and the simplicity of the assay system allow us to modify the functional sequences to recognize different types of biomarkers, thus providing a reference for the identification of various cell subtypes.

Mitochondrial transcription factor A (TFAM) has 5'-deoxyribose phosphate lyase activity in vitro.

Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5'-deoxyribose phosphate (5'dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase ß, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5'dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5'dRp residues at a rate comparable to that of DNA polymerase (pol) ß, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) promotes stress granule formation via YBX1 phosphorylation in ovarian cancer.

APE1 is an essential gene involved in DNA damage repair, the redox regulation of transcriptional factors (TFs) and RNA processing. APE1 overexpression is common in cancers and correlates with poor patient survival. Stress granules (SGs) are phase-separated cytoplasmic assemblies that cells form in response to environmental stresses. Precise regulation of SGs is pivotal to cell survival, whereas their dysregulation is increasingly linked to diseases. Whether APE1 engages in modulating SG dynamics is worthy of investigation. In this study, we demonstrate that APE1 colocalizes with SGs and promotes their formation. Through phosphoproteome profiling, we discover that APE1 significantly alters the phosphorylation landscape of ovarian cancer cells, particularly the phosphoprofile of SG proteins. Notably, APE1 promotes the phosphorylation of Y-Box binding protein 1 (YBX1) at S174 and S176, leading to enhanced SG formation and cell survival. Moreover, expression of the phosphomutant YBX1 S174/176E mimicking hyperphosphorylation in APE1-knockdown cells recovered the impaired SG formation. These findings shed light on the functional importance of APE1 in SG regulation and highlight the importance of YBX1 phosphorylation in SG dynamics.

Construction of an Enzymatically Controlled DNA Nanomachine for One-Step Imaging of Telomerase in Living Cells.

Telomerase is a basic reverse transcriptase that maintains the telomere length in cells, and accurate and specific sensing of telomerase in living cells is critical for medical diagnostics and disease therapeutics. Herein, we demonstrate for the first time the construction of an enzymatically controlled DNA nanomachine with endogenous apurinic/apyrimidinic endonuclease 1 (APE1) as a driving force for one-step imaging of telomerase in living cells. The DNA nanomachine is designed by rational engineering of substrate probes and reporter probes embedded with an enzyme-activatable site (i.e., AP site) and their subsequent assembly on a gold nanoparticle (AuNP). Upon recognition and cleavage of the AP site in the substrate probe by APE1, the loop of the substrate probe unfolds, exposing telomeric primer (TP) with the 3'-OH end. Subsequently, the TP is elongated by telomerase at the 3'-OH end to generate a long telomeric product. The resultant telomeric product acts as a swing arm that can hybridize with a reporter probe to initiate the APE1-powered walking reaction, ultimately generating a significantly enhanced fluorescence signal. Notably, endogenous APE1 is used as the driving force of the DNA nanomachine, avoiding the introduction of exogenous auxiliary cofactors into the cellular microenvironment. Owing to the high kinetics and high amplification efficiency of the APE1-powered DNA nanomachine, this strategy enables one-step sensitive sensing of telomerase in vitro and in vivo. It can successfully discriminate telomerase activity between cancer cells and normal cells, screen telomerase inhibitors, and monitor the variations of telomerase activity in living cells, offering a prospective platform for molecular diagnostics and drug discovery.

Unfilled gaps by polß lead to aberrant ligation by LIG1 at the downstream steps of base excision repair pathway.

Base excision repair (BER) involves the tightly coordinated function of DNA polymerase ß (polß) and DNA ligase I (LIG1) at the downstream steps. Our previous studies emphasize that defective substrate-product channeling, from gap filling by polß to nick sealing by LIG1, can lead to interruptions in repair pathway coordination. Yet, the molecular determinants that dictate accurate BER remains largely unknown. Here, we demonstrate that a lack of gap filling by polß leads to faulty repair events and the formation of deleterious DNA intermediates. We dissect how ribonucleotide challenge and cancer-associated mutations could adversely impact the ability of polß to efficiently fill the one nucleotide gap repair intermediate which subsequently results in gap ligation by LIG1, leading to the formation of single-nucleotide deletion products. Moreover, we demonstrate that LIG1 is not capable of discriminating against nick DNA containing a 3'-ribonucleotide, regardless of base-pairing potential or damage. Finally, AP-Endonuclease 1 (APE1) shows distinct substrate specificity for the exonuclease removal of 3'-mismatched bases and ribonucleotides from nick repair intermediate. Overall, our results reveal that unfilled gaps result in impaired coordination between polß and LIG1, defining a possible type of mutagenic event at the downstream steps where APE1 could provide a proofreading role to maintain BER efficiency.

APE1 inhibition enhances ferroptotic cell death and contributes to hepatocellular carcinoma therapy.

Ferroptosis, a regulated form of cell death triggered by iron-dependent lipid peroxidation, has emerged as a promising therapeutic strategy for cancer treatment, particularly in hepatocellular carcinoma (HCC). However, the mechanisms underlying the regulation of ferroptosis in HCC remain to be unclear. In this study, we have identified a novel regulatory pathway of ferroptosis involving the inhibition of Apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme with dual functions in DNA repair and redox regulation. Our findings demonstrate that inhibition of APE1 leads to the accumulation of lipid peroxidation and enhances ferroptosis in HCC. At the molecular level, the inhibition of APE1 enhances ferroptosis which relies on the redox activity of APE1 through the regulation of the NRF2/SLC7A11/GPX4 axis. We have identified that both genetic and chemical inhibition of APE1 increases AKT oxidation, resulting in an impairment of AKT phosphorylation and activation, which leads to the dephosphorylation and activation of GSK3ß, facilitating the subsequent ubiquitin-proteasome-dependent degradation of NRF2. Consequently, the downregulation of NRF2 suppresses SLC7A11 and GPX4 expression, triggering ferroptosis in HCC cells and providing a potential therapeutic approach for ferroptosis-based therapy in HCC. Overall, our study uncovers a novel role and mechanism of APE1 in the regulation of ferroptosis and highlights the potential of targeting APE1 as a promising therapeutic strategy for HCC and other cancers.

Formation and Properties of DNA Adducts Generated by Reactions of Abasic Sites with 1,2-Aminothiols Including Cysteamine, Cysteine Methyl Ester, and Peptides Containing N-Terminal Cysteine Residues.

The reaction of 1,2-aminothiol groups with aldehyde residues in aqueous solution generates thiazolidine products, and this process has been developed as a catalyst-free click reaction for bioconjugation. The work reported here characterized reactions of the biologically relevant 1,2-aminothiols including cysteamine, cysteine methyl ester, and peptides containing N-terminal cysteine residues with the aldehyde residue of apurinic/apyrimidinic (AP) sites in DNA oligomers. These 1,2-aminothiol-containing compounds rapidly generated adducts with AP sites in single-stranded and double-stranded DNA. NMR and MALDI-TOF-MS analyses provided evidence that the reaction generated a thiazolidine product. Conversion of an AP site to a thiazolidine-AP adduct protected against the rapid cleavage normally induced at AP sites by the endonuclease action of the enzyme APE1 and the AP-lyase activity of the biogenic amine spermine. In the presence of excess 1,2-aminothiols, the thiazolidine-AP adducts underwent slow strand cleavage via a ß-lyase reaction that generated products with 1,2-aminothiol-modified sugar residues on the 3'-end of the strand break. In the absence of excess 1,2-aminothiols, the thiazolidine-AP adducts dissociated to release the parent AP-containing oligonucleotide. The properties of the thiazolidine-AP adducts described here mirror critical properties of SRAP proteins HMCES and YedK that capture AP sites in single-stranded regions of cellular DNA and protect them from cleavage.

Leveraging CRISPR/Cas12 signal amplifier to sensitive detection of apurinic/apyrimidinic endonuclease 1 and high-throughput inhibitor screening.

As an essential protein in DNA repair, apurinic/apyrimidinic endonuclease 1 (APE1) plays multiple critical functions in maintaining homeostasis, making it a significant biomarker and therapeutic target for many disorders. Here, we describe a simple method to detect APE1 based on the Releasing-Extension-Signal amplification Test (REST) strategy that leverages the dsDNA as the activator to fully unlock the trans-cleavage activity of CRISPR/Cas12a. This assay provides a rapid and specific APE1 detection with a detection limit down to 1.05 × 10-5 U/mL. We also combined this method with an automated pipetting platform and a microplate reader for high-throughput screening of potential inhibitors of APE1. Besides, by changing the modification on the probe, the REST strategy was easily repurposed to detect various DNA glycosylases. Taken together, the simplicity and robustness of the method offer a new choice for APE1 detection and inhibitor screening, showing great potential in practical use. Furthermore, the REST strategy devised in this study provides a new example of applying CRISPR/Cas12a signal amplifier to non-nucleic acid biosensing and inhibitor screening, which broadens the CRISPR-Dx toolbox.

AND Logic Gate-Regulated DNAzyme Nanoflower for Monitoring the Activity of Multiple DNA Repair Enzymes.

Despite the progress that has been made in diverse DNA-based nanodevices to in situ monitor the activity of the DNA repair enzymes in living cells, the significance of improving both the sensitivity and specificity has remained largely neglected and understudied. Herein, we propose a regulatable DNA nanodevice to specifically monitor the activity of DNA repair enzymes for early evaluation of cancer mediated by genomic instability. Concretely, an AND logic gate-regulated DNAzyme nanoflower was rationally designed by the self-assembly of the DNA duplex modified with both apurinic/apyrimidinic (AP) site and methyl lesion site. The DNAzyme nanoflower could be reconfigured under the repair of AP sites and O6-methylguanine sites by apurinic/apyrimidinic endonuclease 1 (APE1) and O6-methylguanine methyltransferase (MGMT) to produce a fluorescent signal, realizing the sensitive monitoring of the activity of APE1 and MGMT. Compared to the free DNAzyme duplex, the fluorescent response of the DNAzyme nanoflower increased by 60%, due to the effective enrichment of the DNA probes by the nanoflower structure. More importantly, we have demonstrated that the dual-enzyme activated strategy allows imaging of specific cancer cells in the AND logic gate manner using MCF-7 as a cancer cell model, improving the specificity of cancer cell imaging. This AND logic gate-regulated multifunctional DNAzyme nanoflower provides a simple tool for simultaneously visualizing multiple DNA repair enzymes, holding great potential in early clinical diagnosis and drug discovery.

Interaction of mitoxantrone with abasic sites - DNA strand cleavage and inhibition of apurinic/apyrimidinic endonuclease 1, APE1.

Mitoxantrone (1,4-dihydroxy-5,8-bis[2-(2-hydroxyethylamino)ethylamino]-anthracene-9,10-dione) is a clinically-relevant synthetic anthracenedione that functions as a topoisomerase II poison by trapping DNA double-strand break intermediates. Mitoxantrone binds to DNA via both stacking interactions with DNA bases and hydrogen bonding with the sugar-phosphate backbone. It has been shown that mitoxantrone inhibits apurinic/apyrimidinic (AP) endonuclease 1 (APE1)-catalyzed incision of DNA containing a tetrahydrofuran (THF) moiety and more recently, that mitoxantrone forms Schiff base conjugates at AP sites in DNA. In this study, mitoxantrone-mediated inhibition of APE1 at THF sites was shown to be consistent with preferential binding to, and thermal stabilization of DNA containing a THF site as compared to non-damaged DNA. Investigations into the properties of mitoxantrone at AP and 3' α,ß-unsaturated aldehyde sites demonstrated that in addition to being a potent inhibitor of APE1 at these biologically-relevant substrates (∼ 0.5 µM IC50 on AP site-containing DNA), mitoxantrone also incised AP site-containing DNA by catalyzing ß- and ß/δ-elimination reactions. The efficiency of these reactions to generate the 3' α,ß-unsaturated aldehyde and 3' phosphate products was modulated by DNA structure. Although these cell-free reactions revealed that mitoxantrone can generate 3' phosphates, cells lacking polynucleotide kinase phosphatase did not show increased sensitivity to mitoxantrone treatment. Consistent with its ability to inhibit APE1 activity on DNAs containing either an AP site or a 3' α,ß-unsaturated aldehyde, combined exposures to clinically-relevant concentrations of mitoxantrone and a small molecule APE1 inhibitor revealed additive cytotoxicity. These data suggest that in a cellular context, mitoxantrone may interfere with APE1 DNA repair functions.

In vitro and In vivo evidence demonstrating chronic absence of Ref-1 Cysteine 65 impacts Ref-1 folding configuration, redox signaling, proliferation and metastasis in pancreatic cancer.

Ref-1/APE1 (Redox Effector/Apurinic Endonuclease 1) is a multifunctional enzyme that serves as a redox factor for several transcription factors (TFs), e.g., NF-kB, HIF-1α, which in an oxidized state fail to bind DNA. Conversion of these TFs to a reduced state serves to regulate various biological responses such as cell growth, inflammation, and cellular metabolism. The redox activity involves a thiol exchange reaction for which Cys65 (C65) serves as the nucleophile. Using CRISPR editing in human pancreatic ductal adenocarcinoma (PDAC) cells, we changed C65 to Ala (C65A) in Ref-1 to evaluate alteration of Ref-1 redox dynamics as well as chronic loss of Ref-1 redox activity on cell signaling pathways, specifically those regulated by NF-kB and HIF-1α. The redox activity of Ref-1 requires partial unfolding to expose C65, which is buried in the folded structure. Labeling of Ref-1 with polyethylene glycol-maleimide (PEGm) provides a readout of reduced Cys residues in Ref-1 and thereby an assessment of partial unfolding in Ref-1. In comparing Ref-1WT vs Ref-1C65A cell lines, we found an altered distribution of oxidized versus reduced states of Ref-1. Accordingly, activation of NF-kB and HIF-1α in Ref-1C65A lines was significantly lower compared to Ref-1WT lines. The bioinformatic data revealed significant downregulation of metabolic pathways including OXPHOS in Ref-1C65A expressing clones compared to Ref-1WT line. Ref-1C65A also demonstrated reduced cell proliferation and use of tricarboxylic acid (TCA) substrates compared to Ref-1WT lines. A subcutaneous as well as PDAC orthotopic in vivo model demonstrated a significant reduction in tumor size, weight, and growth in the Ref-1C65A lines compared to the Ref-1WT lines. Moreover, mice implanted with Ref-1C65A redox deficient cells demonstrate significantly reduced metastatic burden to liver and lung compared to mice implanted with Ref-1 redox proficient cells. These results from the current study provide direct evidence that the chronic absence of Cys65 in Ref-1 results in redox inactivity of the protein in human PDAC cells, and subsequent biological results confirm a critical involvement of Ref-1 redox signaling and tumorigenic phenotype.

Thermococcus kodakarensis TK0353 is a novel AP lyase with a new fold.

Hyperthermophilic organisms thrive in extreme environments prone to high levels of DNA damage. Growth at high temperature stimulates DNA base hydrolysis resulting in apurinic/apyrimidinic (AP) sites that destabilize the genome. Organisms across all domains have evolved enzymes to recognize and repair AP sites to maintain genome stability. The hyperthermophilic archaeon Thermococcus kodakarensis encodes several enzymes to repair AP site damage including the essential AP endonuclease TK endonuclease IV. Recently, using functional genomic screening, we discovered a new family of AP lyases typified by TK0353. Here, using biochemistry, structural analysis, and genetic deletion, we have characterized the TK0353 structure and function. TK0353 lacks glycosylase activity on a variety of damaged bases and is therefore either a monofunctional AP lyase or may be a glycosylase-lyase on a yet unidentified substrate. The crystal structure of TK0353 revealed a novel fold, which does not resemble other known DNA repair enzymes. The TK0353 gene is not essential for T. kodakarensis viability presumably because of redundant base excision repair enzymes involved in AP site processing. In summary, TK0353 is a novel AP lyase unique to hyperthermophiles that provides redundant repair activity necessary for genome maintenance.

Coordination between human DNA polymerase ß and apurinic/apyrimidinic endonuclease 1 in the course of DNA repair.

Coordination of enzymatic activities in the course of base excision repair (BER) is essential to ensure complete repair of damaged bases. Two major mechanisms underlying the coordination of BER are known today: the "passing the baton" model and a model of preassembled stable multiprotein repair complexes called "repairosomes." In this work, we aimed to elucidate the coordination between human apurinic/apyrimidinic (AP) endonuclease APE1 and DNA polymerase Polß in BER through studying an impact of APE1 on Polß-catalyzed nucleotide incorporation into different model substrates that mimic different single-strand break (SSB) intermediates arising along the BER pathway. It was found that APE1's impact on separate stages of Polß's catalysis depends on the nature of a DNA substrate. In this complex, APE1 removed 3' blocking groups and corrected Polß-catalyzed DNA synthesis in a coordinated manner. Our findings support the hypothesis that Polß not only can displace APE1 from damaged DNA within the "passing the baton" model but also performs the gap-filling reaction in the ternary complex with APE1 according to the "repairosome" model. Taken together, our results provide new insights into coordination between APE1 and Polß during the BER process.

A New Drug Discovery Platform: Application to DNA Polymerase Eta and Apurinic/Apyrimidinic Endonuclease 1.

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM ("SAR by X-ray Poses Quickly") platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or "undruggable" targets, allows for (i) hit generation; (ii) the mapping of protein-ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.

The DNA glycosylase NEIL2 is protective during SARS-CoV-2 infection.

SARS-CoV-2 infection-induced aggravation of host innate immune response not only causes tissue damage and multiorgan failure in COVID-19 patients but also induces host genome damage and activates DNA damage response pathways. To test whether the compromised DNA repair capacity of individuals modulates the severity of COVID-19 infection, we analyze DNA repair gene expression in publicly available patient datasets and observe a lower level of the DNA glycosylase NEIL2 in the lungs of severely infected COVID-19 patients. This observation of lower NEIL2 levels is further validated in infected patients, hamsters and ACE2 receptor-expressing human A549 (A549-ACE2) cells. Furthermore, delivery of recombinant NEIL2 in A549-ACE2 cells shows decreased expression of proinflammatory genes and viral E-gene, as well as lowers the yield of viral progeny compared to mock-treated cells. Mechanistically, NEIL2 cooperatively binds to the 5'-UTR of SARS-CoV-2 genomic RNA to block viral protein synthesis. Collectively, these data strongly suggest that the maintenance of basal NEIL2 levels is critical for the protective response of hosts to viral infection and disease.

Structural and biochemical insights into NEIL2's preference for abasic sites.

Cellular DNA is subject to damage from a multitude of sources and repair or bypass of sites of damage utilize an array of context or cell cycle dependent systems. The recognition and removal of oxidatively damaged bases is the task of DNA glycosylases from the base excision repair pathway utilizing two structural families that excise base lesions in a wide range of DNA contexts including duplex, single-stranded and bubble structures arising during transcription. The mammalian NEIL2 glycosylase of the Fpg/Nei family excises lesions from each of these DNA contexts favoring the latter two with a preference for oxidized cytosine products and abasic sites. We have determined the first liganded crystal structure of mammalian NEIL2 in complex with an abasic site analog containing DNA duplex at 2.08 Å resolution. Comparison to the unliganded structure revealed a large interdomain conformational shift upon binding the DNA substrate accompanied by local conformational changes in the C-terminal domain zinc finger and N-terminal domain void-filling loop necessary to position the enzyme on the DNA. The detailed biochemical analysis of NEIL2 with an array of oxidized base lesions indicates a significant preference for its lyase activity likely to be paramount when interpreting the biological consequences of variants.

APE1/Ref-1 as a Therapeutic Target for Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammation of the gastrointestinal tract. The prevalence of IBD is increasing with approximately 4.9 million cases reported worldwide. Current therapies are limited due to the severity of side effects and long-term toxicity, therefore, the development of novel IBD treatments is necessitated. Recent findings support apurinic/apyrimidinic endonuclease 1/reduction-oxidation factor 1 (APE1/Ref-1) as a target in many pathological conditions, including inflammatory diseases, where APE1/Ref-1 regulation of crucial transcription factors impacts significant pathways. Thus, a potential target for a novel IBD therapy is the redox activity of the multifunctional protein APE1/Ref-1. This review elaborates on the status of conventional IBD treatments, the role of an APE1/Ref-1 in intestinal inflammation, and the potential of a small molecule inhibitor of APE1/Ref-1 redox activity to modulate inflammation, oxidative stress response, and enteric neuronal damage in IBD.

Levels of APE1 Repair Gene and CLEC4M in Lung Cancer Patients Receiving Cisplatin Chemotherapy.

This study aimed to detect the levels of apurinic/apyrimidinic endonuclease 1 (APE1) gene expression and C-type lectin domain family 4 member M (CLEC4M) and their association with cisplatin chemotherapy in lung cancer patients. Overall, 105 individuals who attended the Al-Amal National Hospital for Cancer Management, Baghdad, Iraq, were enrolled in the study and divided into three equal groups. The groups included the patients newly diagnosed with lung cancer, cancer patients who received cisplatin, and the healthy control group. All study groups were subjected to the sampling of the venous blood for molecular analysis by real-time polymerase chain reaction (RT-PCR) to detect the APE1 gene and enzyme-linked immunosorbent assay (ELISA) for serological testing to measure the concentration of CLEC4M protein. Significantly, the values of both cancer groups were higher than those reported in the control group. The relative index revealed a significant difference in the mean fold change level of APE1 in the newly diagnosed group (3 fold) and cisplatin therapy patients group (2 fold), compared to the control group (P=0.005). No significant differences were detected between the two cancer groups in terms of fold change mean of expression, demographic characteristics, and cancer histological type. Regarding human CLEC4M protein level, cases receiving cisplatin (139.2±25.9) and newly diagnosed patients (331.0±38.1) had a highly significant difference with the control group (100.3±47.5, P<0.001). There was no significant difference between the concentration level of CLEC4M and all parameters in demographic characteristics and cancer histological type. This was the first study to demonstrate that higher expression levels of new APE1, CLEC4M, and glutathione, especially after chemotherapy, are beneficial as diagnostic and prognostic markers for resistance to platinum chemotherapy in Iraqi lung cancer patients.

Unprecedented reactivity of polyamines with aldehydic DNA modifications: structural determinants of reactivity, characterization and enzymatic stability of adducts.

Apurinic/apyrimidinic (AP) sites, 5-formyluracil (fU) and 5-formylcytosine (fC) are abundant DNA modifications that share aldehyde-type reactivity. Here, we demonstrate that polyamines featuring at least one secondary 1,2-diamine fragment in combination with aromatic units form covalent DNA adducts upon reaction with AP sites (with concomitant cleavage of the AP strand), fU and, to a lesser extent, fC residues. Using small-molecule mimics of AP site and fU, we show that reaction of secondary 1,2-diamines with AP sites leads to the formation of unprecedented 3'-tetrahydrofuro[2,3,4-ef]-1,4-diazepane ('ribodiazepane') scaffold, whereas the reaction with fU produces cationic 2,3-dihydro-1,4-diazepinium adducts via uracil ring opening. The reactivity of polyamines towards AP sites versus fU and fC can be tuned by modulating their chemical structure and pH of the reaction medium, enabling up to 20-fold chemoselectivity for AP sites with respect to fU and fC. This reaction is efficient in near-physiological conditions at low-micromolar concentration of polyamines and tolerant to the presence of a large excess of unmodified DNA. Remarkably, 3'-ribodiazepane adducts are chemically stable and resistant to the action of apurinic/apyrimidinic endonuclease 1 (APE1) and tyrosyl-DNA phosphoesterase 1 (TDP1), two DNA repair enzymes known to cleanse a variety of 3' end-blocking DNA lesions.