Apurinic/apyrimidinic Research Articles

A Fluorescent Probe to Measure DNA Damage and Repair.

DNA damage and repair is a fundamental process that plays an important role in cancer treatment. Base excision repair (BER) is a major repair pathway that often leads to drug resistance in DNA-targeted cancer chemotherapy. In order to measure BER, we have developed a near infrared (NIR) fluorescent probe. This probe binds to a key intermediate, termed apurinic/apyrimidinic (AP) site, in the BER pathway where DNA damage and repair occurs. We have developed an assay to show the efficacy of the probe binding to AP sites and have shown that it can distinguish AP sites in DNA extract from chemotherapy treated cells. This probe has potential application in monitoring patient response to chemotherapy and evaluating new drugs in development.

Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes: IDENTIFICATION OF ACCESS RESIDUES

Non-coding apurinic/apyrimidinic (AP) sites are generated at high frequency in genomic DNA via spontaneous hydrolytic, damage-induced or enzyme-mediated base release. AP endonuclease 1 (APE1) is the predominant mammalian enzyme responsible for initiating removal of mutagenic and cytotoxic abasic lesions as part of the base excision repair (BER) pathway. We have examined here the ability of wild-type (WT) and a collection of variant/mutant APE1 proteins to cleave at an AP site within a nucleosome core particle. Our studies indicate that, in comparison to the WT protein and other variant/mutant enzymes, the incision activity of the tumor-associated variant R237C and the rare population variant G241R are uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site. This defect appears to stem from an abnormal interaction of R237C and G241R with abasic DNA substrates, but is not simply due to a DNA binding defect, as the site-specific APE1 mutant Y128A, which displays markedly reduced AP-DNA complex stability, did not exhibit a similar hypersensitivity to nucleosome structures. Notably, this incision defect of R237C and G241R was observed on a pre-assembled DNA glycosylase·AP-DNA complex as well. Our results suggest that the BER enzyme, APE1, has acquired distinct surface residues that permit efficient processing of AP sites within the context of protein-DNA complexes independent of classic chromatin remodeling mechanisms.

Poly(ADP-ribose)polymerase 1 stimulates the AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1.

The influence of poly(ADP-ribose)polymerase 1 (PARP1) on the apurinic/apyrimidinic (AP)-site cleavage activity of tyrosyl-DNA phosphodiesterase 1 (TDP1) and interaction of PARP1 and TDP1 were studied. The efficiency of single or clustered AP-site hydrolysis catalysed by TDP1 was estimated. It was shown that the efficiency of AP-site cleavage increases in the presence of an additional AP-site in the opposite DNA strand depending on its position. PARP1 stimulates TDP1; the stimulation effect was abolished in the presence of NAD(+). The interaction of these two proteins was characterized quantitatively by measuring the dissociation constant for the TDP1-PARP1 complex using fluorescently-labelled proteins. The distance between the N-termini of the proteins within the complex was estimated using FRET. The data obtained suggest that PARP1 and TDP1 bind in an antiparallel orientation; the N-terminus of the former protein interacts with the C-terminal domain of the latter. The functional significance of PARP1 and TDP1 interaction in the process of DNA repair was demonstrated for the first time.

Kinetic analysis of bypass of abasic site by the catalytic core of yeast DNA polymerase eta

Abasic sites (Apurinic/apyrimidinic (AP) sites), produced ∼50,000 times/cell/day, are very blocking and miscoding. To better understand miscoding mechanisms of abasic site for yeast DNA polymerase η, pre-steady-state nucleotide incorporation and LC–MS/MS sequence analysis of extension product were studied using pol ηcore (catalytic core, residues 1–513), which can completely eliminate the potential effects of the C-terminal C2H2 motif of pol η on dNTP incorporation. The extension beyond the abasic site was very inefficient. Compared with incorporation of dCTP opposite G, the incorporation efficiencies opposite abasic site were greatly reduced according to the order of dGTP>dATP>>dCTP and dTTP. Pol ηcore showed no fast burst phase for any incorporation opposite G or abasic site, suggesting that the catalytic step is not faster than the dissociation of polymerase from DNA. LC–MS/MS sequence analysis of extension products showed that 53% products were dGTP misincorporation, 33% were dATP and 14% were -1 frameshift, indicating that Pol ηcore bypasses abasic site by a combined G-rule, A-rule and -1 frameshift deletions. Compared with full-length pol η, pol ηcore relatively reduced the efficiency of incorporation of dCTP opposite G, increased the efficiencies of dNTP incorporation opposite abasic site and the exclusive incorporation of dGTP opposite abasic site, but inhibited the extension beyond abasic site, and increased the priority in extension of A: abasic site relative to G: abasic site. This study provides further understanding in the mutation mechanism of abasic sites for yeast DNA polymerase η.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with apurinic/apyrimidinic sites in DNA

Apurinic/apyrimidinic (AP) sites are some of the most frequent DNA damages and the key intermediates of base excision repair. Certain proteins can interact with the deoxyribose of the AP site to form a Schiff base, which can be stabilized by NaBH4 treatment. Several types of DNA containing the AP site were used to trap proteins in human cell extracts by this method. In the case of single-stranded AP DNA and AP DNA duplex with both 5′ and 3′ dangling ends, the major crosslinking product had an apparent molecular mass of 45kDa. Using peptide mass mapping based on mass spectrometry data, we identified the protein forming this adduct as an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) called “uracil-DNA glycosylase”. GAPDH is a glycolytic enzyme with many additional putative functions, which include interaction with nucleic acids, different DNA damages and DNA repair enzymes. We investigated interaction of GAPDH purified from HeLa cells and rabbit muscles with different AP DNAs. In spite of the ability to form a Schiff-base intermediate with the deoxyribose of the AP site, GAPDH does not display the AP lyase activity. In addition, along with the borohydride-dependent adducts with AP DNAs containing single-stranded regions, GAPDH was also shown to form the stable borohydride-independent crosslinks with these AP DNAs. GAPDH was proven to crosslink preferentially to AP DNAs cleaved via the β-elimination mechanism (spontaneously or by AP lyases) as compared to DNAs containing the intact AP site. The level of GAPDH–AP DNA adduct formation depends on oxidation of the protein SH-groups; disulfide bond reduction in GAPDH leads to the loss of its ability to form the adducts with AP DNA. A possible role of formation of the stable adducts with AP sites by GAPDH is discussed.

Development of APE1 enzymatic DNA repair assays: low APE1 activity is associated with increase lung cancer risk.

The key role of DNA repair in removing DNA damage and minimizing mutations makes it an attractive target for cancer risk assessment and prevention. Here we describe the development of a robust assay for apurinic/apyrimidinic (AP) endonuclease 1 (APE1; APEX1), an essential enzyme involved in the repair of oxidative DNA damage. APE1 DNA repair enzymatic activity was measured in peripheral blood mononuclear cell protein extracts using a radioactivity-based assay, and its association with lung cancer was determined using conditional logistic regression with specimens from a population-based case-control study with 96 lung cancer cases and 96 matched control subjects. The mean APE1 enzyme activity in case patients was 691 [95% confidence interval (CI) = 655-727] units/ng protein, significantly lower than in control subjects (mean = 793, 95% CI = 751-834 units/ng protein, P = 0.0006). The adjusted odds ratio for lung cancer associated with 1 SD (211 units) decrease in APE1 activity was 2.0 (95% CI = 1.3-3.1; P = 0.002). Comparison of radioactivity- and fluorescence-based assays showed that the two are equivalent, indicating no interference by the fluorescent tag. The APE1Asp148Glu SNP was associated neither with APE1 enzyme activity nor with lung cancer risk. Taken together, our results indicate that low APE1 activity is associated with lung cancer risk, consistent with the hypothesis that 'bad DNA repair', rather than 'bad luck', is involved in cancer etiology. Such assays may be useful, along with additional DNA repair biomarkers, for risk assessment of lung cancer and perhaps other cancers, and for selecting individuals to undergo early detection techniques such as low-dose CT.

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins.

Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis

Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3'→5' exonuclease activities. However, it remains unclear whether these enzymes hold 3'-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3'-repair phosphodiesterase and 3'-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5mM MnCl2 at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37 °C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3'-blocking sugar-phosphate and 3'-phosphate moieties at DNA strand breaks with an extremely high efficiency (kcat/KM=440 and 1280 μM(-1)∙min(-1), respectively), while MtbNfo exhibits much lower 3'-repair activities (kcat/KM=0.26 and 0.65 μM(-1)∙min(-1), respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2 to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role in the repair of oxidative DNA damage generated by endogenous and host- imposed factors.

Phase I clinical trial of temozolomide and methoxyamine (TRC-102) in patients with advanced solid tumors.

2558 Background: Temozolomide (TMZ) is an alkylating agent that generates DNA adducts that are repaired by direct DNA and base excision repair mechanisms. Methoxyamine (MX, TRC-102) is a small molecule that potentiates TMZ by binding to apurinic and apyrimidinic sites after removal of N3-methyladenine and N7-methylguanine, inhibiting site recognition of apurinic/apyrimidinic (AP) endonuclease. We conducted a phase I trial to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of intravenous MX when given with oral TMZ. Methods: Patients (pts) with advanced solid tumors, progression on standard treatment, and no CNS involvement were enrolled. A standard 3+3 design was used to assess TMZ (150-200 mg/m2 orally) and MX (15-150 mg/m2 IV). Dose Levels (DL) 1-3: TMZ day 1, MX day 4 in first 14 day cycle (C), TMZ days 1-5, MX day 1 in subsequent 28 day C; DL 4-7: TMZ days 1-5, MX day 1 of 28 day C. DLT period was 42 days for DL 1-3 and 28 days for DL 4-7. Tumor response assessed per REC...

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

Downregulation of hPMC2 imparts chemotherapeutic sensitivity to alkylating agents in breast cancer cells

Triple negative breast cancer cell lines have been reported to be resistant to the cyotoxic effects of temozolomide (TMZ). We have shown previously that a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2) has a role in the repair of estrogen-induced abasic sites. Our present study provides evidence that downregulation of hPMC2 in MDA-MB-231 and MDA-MB-468 breast cancer cells treated with temozolomide (TMZ) decreases cell survival. This increased sensitivity to TMZ is associated with an increase in number of apurinic/apyrimidinic (AP) sites in the DNA. We also show that treatment with another alkylating agent, BCNU, results in an increase in AP sites and decrease in cell survival. Quantification of western blot analyses and immunofluorescence experiments reveal that treatment of hPMC2 downregulated cells with TMZ results in an increase in γ-H2AX levels, suggesting an increase in double strand DNA breaks. The enhancement of DNA double strand breaks in TMZ treated cells upon downregulation of hPCM2 is also revealed by the comet assay. Overall, we provide evidence that downregulation of hPMC2 in breast cancer cells increases cytotoxicity of alkylating agents, representing a novel mechanism of treatment for breast cancer. Our data thus has important clinical implications in the management of breast cancer and brings forth potentially new therapeutic strategies.

The role of His-83 of yeast apurinic/apyrimidinic endonuclease Apn1 in catalytic incision of abasic sites in DNA

BackgroundThe apurinic/apyrimidinic (AP) endonuclease Apn1 from Saccharomyces cerevisiae is a key enzyme involved in the base excision repair (BER) at the cleavage stage of abasic sites (AP sites) in DNA. The crystal structure of Apn1 from S. cerevisiae is unresolved. Based on its high amino acid homology to Escherichia coli Endo IV, His-83 is believed to coordinate one of three Zn2+ ions in Apn1's active site similar to His-69 in Endo IV. Substituting His-83 with Ala is proposed to decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2+ ions involved in enzymatic catalysis. MethodsThe kinetics of recognition, binding, and incision of DNA substrates with the H83A Apn1 mutant was investigated. The stopped-flow method detecting fluorescence intensity changes of 2-aminopurine (2-aPu) was used to monitor the conformational dynamics of DNA at pre-steady-state conditions. ResultsWe found substituting His-83 with Ala influenced catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage. ConclusionsWe consider His-83 properly coordinates the active site Zn2+ ion playing a crucial role in catalytic incision stage. Our data prove suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2+ ions. General significanceOur study provides insights into mechanistic specialty of AP site repair by yeast AP endonuclease Apn1 of Endo IV family, which members are not found in mammals, but are present in many microorganisms. The results will provide useful guidelines for design of new anti-fungal and anti-malarial agents.

Critical determinants for substrate recognition and catalysis in the M. tuberculosis class II AP-endonuclease/3′–5′ exonuclease III

The Mycobacterium tuberculosis AP-endonuclease/3′–5′ exodeoxyribonuclease (MtbXthA) is an important player in DNA base excision repair (BER). We demonstrate that the enzyme has robust apurinic/apyrimidinic (AP) endonuclease activity, 3′–5′ exonuclease, phosphatase, and phosphodiesterase activities. The enzyme functions as an AP-endonuclease at high ionic environments, while the 3′–5′-exonuclease activity is predominant at low ionic environments. Our molecular modelling and mutational experiments show that E57 and D251 are critical for catalysis. Although nicked DNA and gapped DNA are fair substrates of MtbXthA, the gap-size did not affect the excision activity and furthermore, a substrate with a recessed 3′-end is preferred. To understand the determinants of abasic-site recognition, we examined the possible roles of (i) the base opposite the abasic site, (ii) the abasic ribose ring itself, (iii) local distortions in the AP-site, and (iv) conserved residues located near the active site. Our experiments demonstrate that the first three determinants do not play a role in MtbXthA, and in fact the enzyme exhibits robust endonucleolytic activity against single-stranded AP DNA also. Regarding the fourth determinant, it is known that the catalytic-site of AP endonucleases is surrounded by conserved aromatic residues and intriguingly, the exact residues that are directly involved in abasic site recognition vary with the individual proteins. We therefore, used a combination of mutational analysis, kinetic assays, and structure-based modelling, to identify that Y237, supported by Y137, mediates the formation of the MtbXthA–AP-DNA complex and AP-site incision.

AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase β knockout mouse embryonic fibroblasts

Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3′-OH and 5′-deoxyribose phosphate (5′-dRp) termini. DNA polymerase β (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5′-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER.

Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities.

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5′-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5′-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA–R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

Abstract B26: Ape1/Ref-1 directs DNA repair pathway choice, linked to Chk2 signaling, critical for glioblastoma response to chemotherapy

Abstract The predominant human apurinic/apyrimidinic (AP) endonuclease 1, Ape1 (also known as Ref-1) plays a central role in the pathways of base excision repair (BER) of DNA. BER is the major system for correcting the oxidative DNA damage that arises endogenously as a result of metabolism. Spontaneous hydrolytic decay of DNA or DNA glycosylase-mediated hydrolysis of N-glycosyl bonds generates AP sites at an estimated frequency of >10,000 per day in each human cell. If unrepaired, non-instructional AP sites potently block DNA replication and thereby cause toxicity. Thus, the proper repair of AP sites is crucial for maintaining genome stability. Genetic studies in mice support the essential role of Ape1 in cellular viability. In this study, we have explored the molecular details of Ape1-mediated genome maintenance mechanisms, and applied glioblastoma multiforme (GBM) tumor model for investigation of the functional significance of the Ape1-mediated cell responses to chemotherapy. Here we present evidence that Ape1 links to a Chk2-associated tumor suppressor pathway, and is necessary for proper execution of DNA damage-induced checkpoints. Furthermore, our study revealed an unexpected role for Ape1 in BRCA1-mediated homologous recombination (HR) repair of DNA breaks, which is crucial for directing DNA repair pathway choices. The analyses of the TCGA data set for GBM showed that Ape1 and Chk2 are coordinately regulated in glioblastoma multiforme, and increased expression of APEX1 gene coding for Ape1 positively correlates with better survival of glioblastoma patients. GBM is the most common and deadly type of primary brain tumor. The current therapy remains relatively ineffective due to diffuse infiltration and intrinsic resistance of GBM cells. Thus, there is an urgent need for rational treatment protocols for the GBM cure. Our study provides evidence for critical importance of the survival network of Ape1 and Chk2 in GBM response to chemotherapy. Regulation of the equilibrium between Ape1 and Chk2 enables tumor cells switch between DNA repair pathways to favor cell survival. Identification of tumor-specific pre-dominant DNA repair pathways and interruption of such survival networks can be a useful strategy for designing novel therapies for GBM. Citation Format: Thomas Stroebel, Sibylle Madlener, Serkan Tuna, Sarah Vose, Daniella Morse, Bakhos A. Tannous, Tonny Lagerweij, Thomas Wurdinger, Christine Marosi, Irene Slavc, Klemens Vierlinger, Okay Saydam, Brendan D. Price, Bruce Demple, Nurten Saydam. Ape1/Ref-1 directs DNA repair pathway choice, linked to Chk2 signaling, critical for glioblastoma response to chemotherapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B26.

Chemical and toxicological characterization of halobenzoquinones, an emerging class of disinfection byproducts.

Halobenzoquinones (HBQs), a new class of disinfection byproducts (DBPs), occur widely in treated drinking water and recreational water. The main concern regarding human exposure to DBPs stems from epidemiological studies that have consistently linked the consumption of chlorinated drinking water with an increased risk of developing bladder cancer. The U.S. Environmental Protection Agency and Health Canada have set regulations on the amount of DBPs in drinking water to minimize the risk. However, these regulated DBPs do not account for the increased risk of bladder cancer because they have different target organs or lower magnitudes of risk based on animal carcinogenesis studies. Because of the pervasive exposure to DBPs, identification of DBPs relevant to human health has become one of the important research targets to address DBP-associated health concerns. Quantitative structure-toxicity relationship (QSTR) analysis has predicted HBQs to be potential bladder carcinogens. Therefore, this perspective focuses on the chemical and toxicological characterization of HBQs. In vitro cytotoxicity experiments have shown that HBQs induce greater cytotoxicity and/or greater developmental toxicity than most of the regulated DBPs. Cellular mechanistic studies indicate that HBQs are capable of producing reactive oxygen species (ROS) either within cells or in solution, depleting cellular glutathione levels, and influencing cellular antioxidant enzymes, which further induces oxidative stress and oxidative damage to cellular proteins and DNA. Oxidative damage to DNA was demonstrated in the form of significant increases in cellular levels of 8-hydroxydeoxyguanosine (8-OHdG), DNA strand breaks, and apurinic/apyrimidinic (AP) sites. HBQs can also form DNA adducts, affect genome-wide DNA methylation, and inhibit DNA repair enzymes. These findings demonstrate that HBQs are highly cytotoxic and potentially genotoxic and carcinogenic, although in vivo data corroborating this is not available. To fully understand the potential adverse health effects and cancer risk due to HBQ exposure, multidisciplinary research is required regarding human exposure, health risk assessment, and toxicological mechanisms of HBQs.

Inhibition of abasic site cleavage in bubble DNA by multifunctional protein YB-1.

Y-box binding protein 1 (YB-1) is widely known to participate in a multiple DNA and RNA processing events in the living cell. YB-1 is also regarded as a putative component of DNA repair. This possibility is supported by relocalization of YB-1 into the nucleus following genotoxic stress. Increased affinity of YB-1 for damaged DNA, especially in its single-stranded form, and its functional interaction with proteins responsible for the initiation of apurinic/apyrimidinic (AP) site repair, namely, AP endonuclease 1 and DNA glycosylase NEIL1, suggest that YB-1 could be involved in the repair of AP sites as a regulatory protein. Here we show that YB-1 has a significant inhibitory effect on the cleavage of AP sites located in single-stranded DNA and in DNA bubble structures. Such interference may be considered as a possible mechanism to prevent single-stranded intermediates of DNA replication, transcription and repair from being converted into highly genotoxic DNA strand breaks, thus allowing the cell to coordinate different DNA processing mechanisms.

Caenorhabditis elegans EXO-3 contributes to longevity and reproduction: Differential roles in somatic cells and germ cells

Apurinic/apyrimidinic (AP) sites are the major DNA damage generated continuously even under normal conditions, and inhibit DNA replication/transcription. AP endonucleases are ubiquitous enzymes required for the repair of AP sites and 3′ blocking ends, but their physiological roles in multicellular organisms are not fully understood. In this study, we investigated how an AP endonuclease functions in a multicellular organism (Caenorhabditis elegans (C. elegans)). EXO-3 is one of the AP endonucleases in C. elegans. Using an exo-3 mutant worm, we found that deletion of the exo-3 gene caused shortened lifespan in an ung-1-dependent manner. UNG-1 is a uracil DNA glycosylase in C. elegans, and the present finding suggested that UNG-1 is the major producer of AP sites that affects lifespan, and EXO-3 contributes to longevity by completing the repair of uracil. Next we found that the exo-3 gene was abundantly expressed in the gonads, and AP sites in the gonad were efficiently repaired, suggesting that EXO-3 functioned particularly in the gonad. Deletion of the exo-3 gene resulted in a significant decrease in self-brood size. This was rescued by deficiency of NTH-1, which is a bifunctional DNA glycosylase in C. elegans that recognizes oxidative base damage. This result suggested that the major substrate of EXO-3 in the gonad was 3′ blocking end generated by NTH-1, and that EXO-3 played an important role in reproduction. A contribution of EXO-3 to reproduction was also suggested by our finding here that the decrease of self-brood size of the exo-3 mutant became more marked when worms were treated with methyl methanesulfonate (MMS) and sodium bisulfite (NaHSO3). This study demonstrated differential roles of EXO-3 in somatic cells and germ cells.

Suppression of oxidative phosphorylation in mouse embryonic fibroblast cells deficient in apurinic/apyrimidinic endonuclease

The mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair/gene regulatory protein. Decrease of APE1 in cells by inducible shRNA knockdown or by conditional gene knockout caused apoptosis. Here we succeeded in establishing a unique mouse embryonic fibroblast (MEF) line expressing APE1 at a level far lower than those achieved with shRNA knockdown. The cells, named MEF(la) (MEF(lowAPE1)), were hypersensitive to methyl methanesulfonate (MMS), and showed little activity for repairing AP-sites and MMS induced DNA damage. While these results were consistent with the essential role of APE1 in repair of AP sites, the MEF(la) cells grew normally and the basal activation of poly(ADP-ribose) polymerases in MEF(la) was lower than that in the wild-type MEF (MEF(wt)), indicating the low DNA damage stress in MEF(la) under the normal growth condition. Oxidative phosphorylation activity in MEF(la) was lower than in MEF(wt), while the glycolysis rates in MEF(la) were higher than in MEF(wt). In addition, we observed decreased intracellular oxidative stress in MEF(la). These results suggest that cells with low APE1 reversibly suppress mitochondrial respiration and thereby reduce DNA damage stress and increases the cell viability.