Apurinic/apyrimidinic Sites Research Articles

Liquid Chromatography-Nanoelectrospray Ionization-High-Resolution Tandem Mass Spectrometry Analysis of Apurinic/Apyrimidinic Sites in Oral Cell DNA of Cigarette Smokers, e-Cigarette Users, and Nonsmokers.

Cigarette smoking is an established risk factor for oral cancer. The health effects of e-cigarettes are still under investigation but may disturb oral cavity homeostasis and cause lung and cardiovascular diseases. Carcinogens and toxicants in tobacco products and e-cigarettes may damage DNA, resulting in the formation of apurinic/apyrimidinic (AP) sites and initiation of the carcinogenic process. In this study, we optimized a liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method to analyze AP sites in buccal cell DNA of 35 nonsmokers, 30 smokers, and 30 e-cigarette users. AP sites in e-cigarette users (median 3.3 per 107 nts) were significantly lower than in smokers (median 5.7 per 107 nts) and nonsmokers (median 6.0 per 107 nts). AP sites in smokers were not significantly different from nonsmokers (p > 0.05). The e-cigarette vaporizing solvents propylene glycol and glycerin were tested and did not protect against AP site formation in in vitro control and carcinogen exposed rat liver homogenates. However, propylene glycol may inhibit bacteria in oral cells, resulting in reduced inflammation and related effects, and reduced AP site levels in e-cigarette user DNA. This is the first study to examine AP site formation in e-cigarette users and to evaluate AP sites in human oral cell DNA.

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.

Formation and Repair of an Interstrand DNA Cross-Link Arising from a Common Endogenous Lesion.

Interstrand DNA cross-links (ICLs) are cytotoxic because they block the strand separation required for read-out and replication of the genetic information in duplex DNA. The unavoidable formation of ICLs in cellular DNA may contribute to aging, neurodegeneration, and cancer. Here, we describe the formation and properties of a structurally complex ICL derived from an apurinic/apyrimidinic (AP) site, which is one of the most common endogenous lesions in cellular DNA. The results characterize a cross-link arising from aza-Michael addition of the N2-amino group of a guanine residue to the electrophilic sugar remnant generated by spermine-mediated strand cleavage at an AP site in duplex DNA. An α,β-unsaturated iminium ion is the critical intermediate involved in ICL formation. Studies employing the bacteriophage φ29 polymerase provided evidence that this ICL can block critical DNA transactions that require strand separation. The results of biochemical studies suggest that this complex strand break/ICL might be repaired by a simple mechanism in which the 3'-exonuclease action of the enzyme apurinic/apyrimidinic endonuclease (APE1) unhooks the cross-link to initiate repair via the single-strand break repair pathway.

Identification of a novel bifunctional uracil DNA glycosylase from Thermococcus barophilus Ch5.

Genomes of hyperthermophiles are facing a severe challenge due to increased deamination rates of cytosine induced by high temperature, which could be counteracted by base excision repair mediated by uracil DNA glycosylase (UDG) or other repair pathways. Our previous work has shown that the two UDGs (Tba UDG247 and Tba UDG194) encoded by the genome of the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 can remove uracil from DNA at high temperature. Herein, we provide evidence that Tba UDG247 is a novel bifunctional glycosylase which can excise uracil from DNA and further cleave the phosphodiester bo nd of the generated apurinic/apyrimidinic (AP) site, which has never been described to date. In addition to cleaving uracil-containing DNA, Tba UDG247 can also cleave AP-containing ssDNA although at lower efficiency, thereby suggesting that the enzyme might be involved in repair of AP site in DNA. Kinetic analyses showed that Tba UDG247 displays a faster rate for uracil excision than for AP cleavage, thus suggesting that cleaving AP site by the enzyme is a rate-limiting step for its bifunctionality. Phylogenetic analysis showed that Tba UDG247 is clustered on a separate branch distant from all the reported UDGs. Overall, we designated Tba UDG247 as the prototype of a novel family of bifunctional UDGs. KEY POINTS: We first reported a novel DNA glycosylase with bifunctionality. Tba UDG247 possesses an AP lyase activity.

A Single Gene-Mediated Perturbation of the Human Genome Leads to Malignant Transformation in a Murine Transgenic Model

We hypothesized that induction or activation of a gene that directly affects DNA integrity can provide a tool for ongoing genomic changes in a cell and may eventually lead to oncogenesis. In the human genome, apurinic/apyrimidinic (AP) sites are created by number of external and internal factors including exposure to chemicals, UV radiation, DNA damaging agents, reactive oxygen species (ROS), DNA glycosylases and/or spontaneous hydrolysis by other mechanisms. AP sites are pre-mutagenic lesions that can also affect normal DNA replication. An evolutionarily conserved mechanism “base excision repair (BER)” has been established to identify and repair such sites. As part of BER, AP nucleases cleave the phosphodiester backbone at 5' to the AP site, prior to its repair. Although APEX 1 and 2 are the nucleases required for this repair, their hyperactivity has been connected with increased DNA breaks. We have previously observed that over expression of either APEX 1 or APEX 2 leads to malignant transformation with number of genomic changes including copy number alterations, single nucleotide variants (SNVs) and structural changes.In this study, we investigated if transgenic overexpression of APEX2 in B cells in mice will cause tumorogenesis. We specifically overexpressed APEX2 in B cells by generating Tg,CD19Cre+/-,APEX2+/- mouse lines and used Tg,CD19Cre+/- mouse (siblings of trail group) as control. The expression of APEX2 was detected by immunohistochemical (IHC) staining, which confirmed specific expression of APEX2 in lymph nodes. We next evaluated impact of APEX2 expression on B cell genome. We isolated B220+ B cells from spleen of 8 weeks old mice, and identified both increased single strand break (SSB) and double strand breaks (DSB). After 45 weeks of age and at variable times thereafter, 40% (23/58) of Tg,CD19Cre+/-,APEX2+/- mice showed enlargement of the lymph nodes and spleen. Histology showed effacement of normal tissue architecture by a nodular or diffuse population of atypical lymphoid cells in interfollicular areas. A distinct pattern of B220+BCL6+CD5- B-cell lymphomas were identified after IHC staining, which resemble human diffuse large B-cell lymphoma (DLBCL). To investigate the changes at genomic level, we performed whole genome sequencing of tumor tissues from 3 mice as well as heart tissues (with lowest APEX2 expression) from the same mice to be used as controls. From single nucleotide varients (SNVs) calling, we observed average of 24,388 SNVs in the tumor with acquisition of new copy number changes and significant number of structural variants including average of 43 translocations, 51 deletions, 21 duplications and 24 inversion. Indel frequency was variable with average of 348. We observed mutational hotspots on chr6, chr12 and chr16. Variant allele frequency (VAF) data shows clonality of chr11 related mutations. We observed most frequent T>G, and then T>C mutations in all 3 samples. Interestingly, the mutational signature analysis showed signature 17 accounting for an average of 60% of mutations followed by signature 9. According to the mutational signature profile of different diseases, both signature 9 and 17 are observed in human B cell lymphoma.To understand the genesis of the malignant transformation, we scarified Tg,CD19Cre+/-,APEX2+/- mice and control Tg,CD19Cre+/- mouse at 25 weeks after birth to see the pre-malignant changes in lymphoid organs. We observed enlarged and increased follicle numbers in lymph nodes and in spleen in Tg,CD19Cre+/-,APEX2+/- mice compared to control. A single cell sequencing is underway to detect clonal changes at an early stage.Our results demonstrate a single DNA-repair related gene responsible for inducing genomic instability with malignant transformation providing a model to study oncogenesis as well as possible preventative measures. DisclosuresAnderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; MedImmune: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Evolutionary Origins of DNA Repair Pathways: Role of Oxygen Catastrophe in the Emergence of DNA Glycosylases

It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the major factor affecting LUCA’s genome integrity. Cosmic radiation due to Earth’s weak magnetic field and alkylating metabolic radicals added to these threats. Here, we propose that ancient forms of life had only two distinct repair mechanisms: versatile apurinic/apyrimidinic (AP) endonucleases to cope with both AP sites and deaminated residues, and enzymes catalyzing the direct reversal of UV and alkylation damage. The absence of uracil–DNA N-glycosylases in some Archaea, together with the presence of an AP endonuclease, which can cleave uracil-containing DNA, suggests that the AP endonuclease-initiated nucleotide incision repair (NIR) pathway evolved independently from DNA glycosylase-mediated base excision repair. NIR may be a relic that appeared in an early thermophilic ancestor to counteract spontaneous DNA damage. We hypothesize that a rise in the oxygen level in the Earth’s atmosphere ~2 Ga triggered the narrow specialization of AP endonucleases and DNA glycosylases to cope efficiently with a widened array of oxidative base damage and complex DNA lesions.

Cytotoxicity and genotoxicity of coated-gold nanoparticles on freshwater algae Pseudokirchneriella subcapitata.

Gold engineered nanoparticles (nAu) are increasingly detected in ecosystems, and this raises the need to establish their potential effects on aquatic organisms. Herein, cytotoxic and genotoxic effects of branched polyethylenimine (BPEI)- and citrate (cit)-coated nAu (5, 20, and 40 nm) on algae Pseudokirchneriella subcapitata were evaluated. The apical biological endpoints: growth inhibition and chlorophyll a (Chl a) content were investigated at 62.5-1000 µg/L over 168 h. In addition, the apurinic/apyrimidinic (AP) sites, randomly amplified polymorphic deoxyribonucleic acid (RAPD) profiles, and genomic template stability (GTS) were assessed to determine the genotoxic effects of nAu. The results show algal growth inhibition at 5 nm BPEI-nAu up to 96 h, and thereafter cell recovery except at the highest concentration of 1000 µg/L. Insignificant growth reduction for cit-nAu (all sizes), as well as 20 and 40 nm BPEI-nAu, was observed over 96 h, but growth promotion was apparent at all exposures thereafter except for 40 nm BPEI-nAu at 250 µg/L. A decrease in Chl a content following exposure to 5 nm BPEI-nAu at 1000 µg/L corresponded to significant algal growth reduction. In genotoxicity studies, a significant increase in AP sites content was observed relative to the control - an indication of nAu ability to induce genotoxic effects irrespective of their size and coating type. For 5 nm- and 20 nm-sized nAu for both coating types and exposure concentrations no differences in AP sites content were observed after 72 and 168 h. However, a significant reduction in AP sites was observed following algae exposure to 40 nm-sized nAu (irrespective of coating type and exposure concentration) at 168 h compared to 72 h. Thus, AP sites results at 40 nm-size suggest likely DNA damage recovery over a longer exposure period. The findings on AP sites content showed a good correlation with an increase in genome template stability and growth promotion observed after 168 h. In addition, RAPD profiles demonstrated that nAu can induce DNA damage and/or DNA mutation to P. subcapitata as evidenced by the appearance and/or disappearance of normal bands compared to the controls. Therefore, genotoxicity results revealed significant toxicity of nAu to algae at the molecular level although no apparent effects were detectable at the morphological level. Overall, findings herein indicate that long-term exposure of P. subcapitata to low concentrations of nAu may cause undesirable sub-lethal ecological effects.

Simple Mix-and-Read Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Sensitive Detection of DNA Glycosylase.

Human 8-oxoguanine DNA glycosylase (hOGG1) can initiate base excision repair of genomic 8-oxoguanine (8-oxoG), and it can locate and remove damaged 8-oxoG through extrusion and excision. Sensitive detection of hOGG1 is critical for clinical diagnosis. Herein, we develop a simple mix-and-read assay for the sensitive detection of DNA glycosylase using multiple cyclic enzymatic repairing amplification. The hOGG1 can excise the 8-oxoG base of the DNA substrate to produce an apurinic/apyrimidinic (AP) site, and then, the AP site can be cleaved by apurinic/apyrimidic endonuclease 1 (APE1), producing the substrate fragment with a free 3'-OH terminus. Subsequently, the substrate fragment can initiate cyclic enzymatic repairing amplification, generating two triggers. The resultant two triggers can function as the primers to induce three cyclic enzymatic repairing amplification, respectively, producing more and more triggers. We experimentally verify the occurrence of each cyclic enzymatic repairing amplification and uracil DNA glycosylase (UDG)-mediated exponential amplification. The amplification products can be simply detected using SYBR Green II as the fluorescent dye. This mix-and-read assay is very simple and rapid (within 40 min) without the requirement of any extra primers and modification/separation steps. This method can sensitively measure hOGG1 with a detection limit of 2.97 × 10-8 U/μL, and it can be applied for the screening of inhibitors and the monitoring of cellular hOGG1 activity at the single-cell level, providing an adaptive and flexible tool for clinical diagnosis and drug discovery.

How (5'S) and (5'R) 5',8-Cyclo-2'-Deoxypurines Affect Base Excision Repair of Clustered DNA Damage in Nuclear Extracts of xrs5 Cells? A Biochemical Study.

The clustered DNA lesions (CDLs) are a characteristic feature of ionizing radiation’s impact on the human genetic material. CDLs impair the efficiency of cellular repair machinery, especially base excision repair (BER). When CDLs contain a lesion repaired by BER (e.g., apurinic/apyrimidinic (AP) sites) and a bulkier 5′,8-cyclo-2′-deoxypurine (cdPu), which is not a substrate for BER, the repair efficiency of the first one may be affected. The cdPus’ influence on the efficiency of nuclear BER in xrs5 cells have been investigated using synthetic oligonucleotides with bi-stranded CDL (containing (5′S) 5′,8-cyclo-2′-deoxyadenosine (ScdA), (5′R) 5′,8-cyclo-2′-deoxyadenosine (RcdA), (5′S) 5′,8-cyclo-2′-deoxyguanosine (ScdG) or (5′R) 5′,8-cyclo-2′-deoxyguanosine (RcdG) in one strand and an AP site in the other strand at different interlesion distances). Here, for the first time, the impact of ScdG and RcdG was experimentally tested in the context of nuclear BER. This study shows that the presence of RcdA inhibits BER more than ScdA; however, ScdG decreases repair level more than RcdG. Moreover, AP sites located ≤10 base pairs to the cdPu on its 5′-end side were repaired less efficiently than AP sites located ≤10 base pairs on the 3′-end side of cdPu. The strand with an AP site placed opposite cdPu or one base in the 5′-end direction was not reconstituted for cdA nor cdG. CdPus affect the repair of the other lesion within the CDL. It may translate to a prolonged lifetime of unrepaired lesions leading to mutations and impaired cellular processes. Therefore, future research should focus on exploring this subject in more detail.

Genetic Polymorphisms in DNA Repair Gene APE1/Ref-1 and the Risk of Neural Tube Defects in a High-Risk Area of China.

Apurinic/apyrimidinic endonuclease 1/redox-factor 1 (APE1/Ref-1) gene encodes a multifunctional protein involved in the DNA base excision repair (BER) pathway, which initiates repair of apurinic/apyrimidinic (AP) sites in DNA by catalyzing hydrolytic incision of the phosphodiester backbone. APE1/Ref-1 polymorphisms are related to the occurrence of neural tube defects (NTDs), but the association between APE1/Ref-1 polymorphisms and NTDs is not reported in Chinese Han population. The aim of the present study was to evaluate the association of APE1/Ref-1 polymorphism and the risk of NTD occurrence for Han population in a high-risk area of China. APE1/Ref-1 genotypes were determined by iPLEX Gold SNP genotyping. AP sites and folate level of brain tissues were measured. The results showed that three polymorphisms (rs3136817, rs77794916, and rs1760944) of APE1/Ref-1 were statistically associated with NTD subtypes. Allele C of rs3136817, allele T of rs77794916, and allele G of rs1760944 were associated with an increased risk for encephalocele (OR = 2.52, 95% CI [1.25-5.07], P < 0.01; OR = 1.80, 95% CI [1.04-3.12], P = 0.04; and OR = 1.96, 95% CI [1.12-3.45], P = 0.02), compared with those harboring the alleles T, C, and T, respectively. The folate level in NTDs was lower than that in controls. DNA AP sites in the encephalocele were significantly higher than the control (P < 0.01). The three polymorphisms of APE1/Ref-1 were significantly related to NTD occurrence, which indicated that APE1/Ref-1 might be a potential genetic risk factor for encephalocele in a high-risk area of NTDs in China.

Mitochondrial apurinic/apyrimidinic endonuclease Apn1 is not critical for the completion of the Plasmodium berghei life cycle

Mitochondrion is an essential organelle in malaria parasite and its DNA must be maintained for optimal function during its complex life cycle. Base excision repair is one of the major pathways by which this is achieved. Apurinic/apyrimidinic (AP) endonucleases are important components of this pathway as they create a nick at the 5′-phosphodiester bond in the AP site and generate free 5′-phosphate and 3′-hydroxyl groups. Two class II AP endonucleases (Apn1 and Ape1) have been annotated in the Plasmodium berghei genome. Using reverse genetic approaches, we provide direct evidence that Apn1 is exclusively localized to the mitochondria of P. berghei. Surprisingly, our gene deletion study revealed a completely dispensable role of Apn1 for the entirety of the P. berghei life cycle. Apn1− parasites were found to successfully grow in the blood. They were transmitted normally to the mosquito midguts and salivary glands. Sporozoites obtained from the salivary glands were infective and achieved similar patency as WT. Our results help emphasize the non-availability of this enzyme as a plausible drug target. We also emphasize the importance of genetic validation of antimalarial drug targets before furthering them down the drug discovery pipeline.

Characterization of Recombinant Endonuclease IV from Mycobacterium tuberculosis

Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role in protecting mycobacterial DNA from oxidative stress. Mycobacterial endonuclease IV substantially differs from its homologs found in Escherichia coli and other proteobacteria in a number of conserved positions important for DNA binding and AP site recognition. The M. tuberculosis end gene was cloned, and recombinant MtbEnd purified and characterized. The protein efficiently hydrolyzed DNA at the natural AP site and its 1'-deoxy analog in the presence of divalent cations, of which Ca^(2+), Mn^(2+), and Co^(2+) supported the highest activity. Exonuclease activity was not detected in MtbEnt preparations. The pH optimum was estimated at 7.0-8.0; the ionic strength optimum, at ~50 mM NaCl. Enzymatic activity of MtbEnd was suppressed in the presence of methoxyamine, a chemotherapeutic agent that modifies AP sites. Based on the results, MtbEnd was assumed to provide a possible target for new anti-tuberculosis drugs.

Activity of Human Apurinic/Apyrimidinic Endonuclease APE1 Toward Damaged DNA and Native RNA With Non-canonical Structures.

The primary role of apurinic/apyrimidinic (AP) endonuclease APE1 in human cells is the cleavage of the sugar phosphate backbone 5′ to an AP site in DNA to produce a single-strand break with a 5′-deoxyribose phosphate and 3′-hydroxyl end groups. APE1 can also recognize and incise some damaged or modified nucleotides and possesses some minor activities: 3′–5′ exonuclease, 3′-phosphodiesterase, 3′-phosphatase, and RNase H. A molecular explanation for the discrimination of structurally different substrates by the single active site of the enzyme remains elusive. Here, we report a mechanism of target nucleotide recognition by APE1 as revealed by the results of an analysis of the APE1 process involving damaged DNA and native RNA substrates with non-canonical structures. The mechanism responsible for substrate specificity proved to be directly related to the ability of a target nucleotide to get into the active site of APE1 in response to an enzyme-induced DNA distortion.

Integration of magnetic separation and real-time ligation chain reaction for detection of uracil-DNA glycosylase.

Uracil-DNA glycosylase (UDG) is a protein enzyme that initiates the base excision repair pathway for maintaining genome stability. Sensitive detection of UDG activity is important in the study of many biochemical processes and clinical applications. Here, a method for detecting UDG is proposed by integrating magnetic separation and real-time ligation chain reaction (LCR). First, a DNA substrate containing uracil base is designed to be conjugated to the magnetic beads. By introducing a DNA complementary to the DNA substrate, the uracil base is recognized and removed by UDG to form an apurinic/apyrimidinic (AP) site. The DNA substrate is then cut off from the AP site by endonuclease IV, releasing a single-strand DNA (ssDNA). After magnetic separation, the ssDNA is retained in the supernatant and then detected by real-time LCR. The linear range of the method is 5 × 10-4 to 5U/mL with four orders of magnitude, and the detection limit is 2.7 × 10-4 U/mL. In the assay, ssDNA template obtained through magnetic separation can prevent other DNA from affecting the subsequent LCR amplification reaction, which provides a simple, sensitive, specific, and universal way to detect UDG and other repair enzymes. Furthermore, the real-time LCR enables the amplification reaction and fluorescence detection simultaneously, which simplifies the operation, avoids post-contamination, and widens the dynamic range. Therefore, the integration of magnetic separation and real-time LCR opens a new avenue for the detection of UDG and other DNA repair enzymes.

A Multi-Endpoint Approach to Base Excision Repair Incision Activity Augmented by PARylation and DNA Damage Levels in Mice: Impact of Sex and Age.

Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2’-deoxyguanosine (8-oxodG), 5-hydroxy-2’-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.

Mass Spectrometric Quantitation of Apurinic/Apyrimidinic Sites in Tissue DNA of Rats Exposed to Tobacco-Specific Nitrosamines and in Lung and Leukocyte DNA of Cigarette Smokers and Nonsmokers.

Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl adducts. Among these are adducts such as 7-POB-deoxyguanosine (N7POBdG) which can lead to apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. In this study, we used a recently developed highly sensitive mass spectrometric method to quantitate AP sites by derivatization with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA) (detection limit, 2 AP sites per 108 nucleotides). AP sites were quantified in DNA isolated from tissues of rats treated with NNN and NNK and from human lung tissue and leukocytes of cigarette smokers and nonsmokers. Rats treated with 5 or 21 mg/kg bw NNK for 4 days by s.c. injection had 2-6 and 2-17 times more AP sites than controls in liver and lung DNA (p < 0.05). Increases in AP sites were also found in liver DNA of rats exposed for 10 and 30 weeks (p < 0.05) but not for 50 and 70 weeks to 5 ppm of NNK in their drinking water. Levels of N7POBG were significantly correlated with AP sites in rats treated with NNK. In rats treated with 14 ppm (S)-NNN in their drinking water for 10 weeks, increased AP site formation compared to controls was observed in oral and nasal respiratory mucosa DNA (p < 0.05). No significant increase in AP sites was found in human lung and leukocyte DNA of cigarette smokers compared to nonsmokers, although AP sites in leukocyte DNA were significantly correlated with urinary levels of the NNK metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). This is the first study to use mass spectrometry based methods to examine AP site formation by carcinogenic tobacco-specific nitrosamines in laboratory animals and to evaluate AP sites in DNA of smokers and nonsmokers.

Structural and Functional Characterization of a Unique AP Endonuclease From Deinococcus radiodurans.

Various endogenous and exogenous agents cause DNA damage, including apurinic/apyrimidinic (AP) sites. Due to their cytotoxic effects, AP sites are usually cleaved by AP endonuclease through the base excision repair (BER) pathway. Deinococcus radiodurans, an extraordinary radiation-resistant bacterium, is known as an ideal model organism for elucidating DNA repair processes. Here, we have investigated a unique AP endonuclease (DrXth) from D. radiodurans and found that it possesses AP endonuclease, 3′-phosphodiesterase, 3′-phosphatase, and 3′–5′ exonuclease but has no nucleotide incision repair (NIR) activity. We also found that Mg2+ and Mn2+ were the preferred divalent metals for endonuclease and exonuclease activities, respectively. In addition, DrXth were crystallized and the crystals diffracted to 1.5 Å. Structural and biochemical analyses demonstrated that residue Gly198 is the key residue involved in the substrate DNA binding and cleavage. Deletion of the drxth gene in D. radiodurans caused elevated sensitivity to DNA damage agents and increased spontaneous mutation frequency. Overall, our results indicate that DrXth is an important AP endonuclease involved in BER pathway and functions in conjunction with other DNA repair enzymes to maintain the genome stability.

Endogenous oxidized DNA bases and APE1 regulate the formation of G-quadruplex structures in the genome

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.

An Assay for the Activity of Base Excision Repair Enzymes in Cellular Extracts Using Fluorescent DNA Probes.

Damaged DNA bases are removed by the base excision repair (BER) mechanism. This enzymatic process begins with the action of one of DNA glycosylases, which recognize damaged DNA bases and remove them by hydrolyzing N-glycosidic bonds with the formation of apurinic/apyrimidinic (AP) sites. Apurinic/apyrimidinic endonuclease 1 (APE1) hydrolyzes the phosphodiester bond on the 5'-side of the AP site with generation of the single-strand DNA break. A decrease in the functional activity of BER enzymes is associated with the increased risk of cardiovascular, neurodegenerative, and oncological diseases. In this work, we developed a fluorescence method for measuring the activity of key human DNA glycosylases and AP endonuclease in cell extracts. The efficacy of fluorescent DNA probes was tested using purified enzymes; the most efficient probes were tested in the enzymatic activity assays in the extracts of A549, MCF7, HeLa, WT-7, HEK293T, and HKC8 cells. The activity of enzymes responsible for the repair of AP sites and removal of uracil and 5,6-dihydrouracil residues was higher in cancer cell lines as compared to the normal HKC8 human kidney cell line.

Abstract TMP35: Neural Recovery Following Cerebral Ischemia is Exacerbated by Loss of DNA Polymerase-Beta

Oxidative DNA damage (ODD) after cerebral ischemia/reperfusion injury occurs rapidly and has been implicated in the patholgenesis of ischemic injury. Ischemic ODD in the brain are primarly characterized as apurinic/apyrimidinic (AP) sites and single strand breaks (SSB). Following ODD, key enzymes in the base excision-repair (BER) pathway including AP endonuclease-1 (APE1) and the DNA polymerase-beta (PolyB) are critical to repair AP sites and SSB, and to mitigate cellular damage. Global knockout of either APE1 or PolyB leads to embryonic lethality. We have previously demonstrated that conditional deletion of APE1 in the adult heightens sensitivity to ischemic injury. PolyB is a highly inducible gene, and its expression is increased after sublethal brain injury, yet the contribution of PolyB to ischemic brain recovery remains unknown. PolyB conditional knockout (cKO) mice were generated by subjecting PolyB-CAG-CreER mice to tamoxifen administration for 5 days. Transient middle cerebral artery occlusion (tMCAO) was induced for 45 min following STAIR guidelines, and behavioral recovery and grey/white matter injury were assessed by over 35 days after ischemia. Cortical action field potentials (CAFP) and compound action potentials (CAP) were used to assess functional integrity of grey and white matter. PolyB cKO mice were significantly impaired up to 35 days after ischemic injury compared to WT mice, displaying exacerbated neuronal tissue loss, extensive demyelination in the CC/EC, and diminished CAFP and CAP compared to WT mice. In addition, accumulation of AP sites and SSBs were evident in the ischemic PolyB cKO. These results were compared to to APE1 cKO ischemic outcomes. PolyB cKO was associated with PARP1 activation, NAD + depletion, necrotic neuronal cell death, and neuronal release of HMGB1, and PJ34, a specific PARP-1 inhibitor, significantly improved stroke outcomes in PolyB cKO mice. Along with APE1, PolyB plays an essential role in ischemic brain recovery in both grey and white matter. However, the molecular mechanisms between exacerbation of injury in ischemic neurons by PolyB vs APE1 may be distinct. DNA base excision repair pathway may potentially serve as a therapeutic target for long-term neurological recovery after stroke.