AP Sites In DNA Research Articles

Role of the nucleotide excision repair pathway proteins (UvrB and UvrD2) in recycling UdgB, a base excision repair enzyme in Mycobacterium smegmatis

Cross-talks between DNA repair pathways are emerging as a crucial strategy in the maintenance of the genomic integrity. A double-stranded (ds) DNA specific DNA glycosylase, UdgB is known to excise uracil, hypoxanthine and ethenocytosine. We earlier showed that Mycobacterium smegmatis (Msm) UdgB stays back on the AP-sites it generates in the DNA upon excision of the damaged bases. Here, we show that in an Msm strain deleted for a nucleotide excision repair (NER) protein, UvrB (uvrB–), UdgB expression is toxic, and its deletion from the genome (udgB–) rescues the strain from the genotoxic stress. However, UdgB bound AP-site is not a direct substrate for NER in vitro. We show that UvrD2 and UvrB, known helicases with single-stranded (ss) DNA translocase activity, facilitate recycling of UdgB from AP-DNA. Our studies reveal that the helicases play an important role in exposing the AP-sites in DNA and make them available for further repair.

Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity.

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg2+-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine–purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg2+ ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine–purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg^(2+)-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg^(2+) ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

Characterization and application of a family B DNA polymerase from the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans

Thermococcus gammatolerans is anaerobic euryarchaeon which grows optimally at 88 °C and its genome encodes a family B DNA polymerase (Tga PolB). Herein, we cloned the gene of Tga PolB, expressed and purified the gene product, and characterized the enzyme biochemically. The recombinant Tga PolB can efficiently synthesize DNA at high temperature, and retain 93% activity after heated at 95 °C for 1.0 h, suggesting that the enzyme is thermostable. Furthermore, the optimal pH for the enzyme activity was measured to be 7.0–9.0. Tga PolB activity is dependent on a divalent cation, among which magnesium ion is optimal. NaCl at low concentration stimulates the enzyme activity but at high concentration inhibits enzyme activity. Interestingly, Tga PolB is able to efficiently bypass uracil in DNA, which is distinct from other archaeal family B DNA pols. By contrast, Tga PolB is halted by an AP site in DNA, as observed in other archaeal family B DNA polymerases. Furthermore, Tga PolB extends the mismatched ends with reduced efficiencies. The enzyme possesses 3′-5′ exonuclease activity and this activity is inhibited by dNTPs. The DNA binding assays showed that Tga PolB can efficiently bind to ssDNA and primed DNA, and have a marked preference for primed DNA. Last, Tga PolB can be used in routine PCR.

Ultrafast Oxime Formation Enables Efficient Fluorescence Light-up Measurement of DNA Base Excision.

DNA glycosylases constitute a biologically and biomedically important group of DNA repair enzymes responsible for initiating base excision repair (BER). Measuring their activities can be useful for studying the mechanisms DNA damage and repair and for practical applications in cancer diagnosis and drug screening. Previous fluorescence methods for assaying DNA glycosylases are often complex and/or limited in scope to a single enzyme type. Here we report a universal base excision reporter (UBER) fluorescence probe design that implements an unprecedentedly rapid oxime reaction (>150 M-1 s-1) with high specificity for the abasic (AP) site of DNA. The molecular rotor design achieves a robust >250-500-fold increase in fluorescence upon reaction with AP sites in DNA. By using the fluorescence reporter in concert with specific DNA lesion-containing substrates, the UBER probe can be used in a coupled assay in principle with any DNA glycosylase. We demonstrate the utility of the UBER probe by assaying five different glycosylases in real time as well as profiling glycosylase activity in cell lysates. We anticipate that the UBER probe will be of considerable utility to researchers studying DNA repair biology owing to its high level of generalizability, ease of use, and compatibility with biologically derived samples.

The impact of single-nucleotide polymorphisms of human apurinic/apyrimidinic endonuclease 1 on specific DNA binding and catalysis

Human apurinic/apyrimidinic (AP) endonuclease APE1 is a crucial enzyme of the base excision repair (BER) pathway, which is in charge of recognition and initiation of removal of AP-sites in DNA. It is known that some single-nucleotide polymorphism (SNP) variants of APE1 have a reduced activity as compared to wild-type APE1. It has been hypothesized that genetic variation in APE1 might be responsible for an increased risk of some types of cancer. In the present work, analysis of SNPs of the APE1 gene was performed to select the set of variants having substitutions of amino acid residues on the surface of the enzyme globule and in the DNA-binding site, thereby affecting protein–protein interactions or the catalytic reaction, respectively. For seven APE1 variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S), conformational dynamics and catalytic activities were examined. The conformational changes in the molecules of APE1 variants and in a DNA substrate were recorded as fluorescence changes of Trp and 2-aminopurine residues, respectively, using the stopped-flow technique. The results made it possible to determine the kinetic mechanism underlying the interactions of the APE1 variants with DNA substrates, to calculate the rate constants of the elementary stages, and to identify the stages of the process affected by mutation.

Interaction of Functionalized Naphthalenophanes with Abasic Sites in DNA: DNA Cleavage, DNA Cleavage Inhibition, and Formation of Ligand-DNA Adducts.

Ligands interacting with abasic (AP) sites in DNA may generate roadblocks in base-excision DNA repair (BER) due to indirect inhibition of DNA repair enzymes (e.g., APE1) and/or formation of toxic byproducts, resulting from ligand-induced strand cleavage or covalent cross-links. Herein, a series of 12 putative AP-site ligands, sharing the common naphthalenophane scaffold, but endowed with a variety of substituents, have been prepared and systematically studied. The results demonstrate that most naphthalenophanes bind to AP sites in DNA and inhibit the APE1-induced hydrolysis of the latter in vitro. Remarkably, their APE1 inhibitory activity, as characterized by IC50 and KI values, can be directly related to their affinity and selectivity to AP sites, as assessed by means of fluorescence melting experiments. On the other hand, the molecular design of naphthalenophanes has a crucial influence on their intrinsic AP-site cleavage activity (i.e., ligand-catalyzed β- and β,δ-elimination reactions at the AP site), as illustrated by the compounds either having an exceptionally high AP-site cleavage activity (e.g., 2,7-BisNP-S, 125-fold more efficacious than spermine) or being totally devoid of this activity (four compounds). Finally, the unprecedented formation of a stable covalent DNA adduct upon reaction of one ligand (2,7-BisNP-NH) with its own product of the AP-site cleavage is revealed.

Ultrasensitive recognition of AP sites in DNA at the single-cell level: one molecular rotor sequentially self-regulated to form multiple different stable conformations.

The AP site is a primary form of DNA damage. Its presence alters the genetic structure and eventually causes malignant diseases. AP sites generally present a high-speed dynamic change in the DNA sequence. Thus, precisely recognizing AP sites is difficult, especially at the single-cell level. To address this issue, we provide a broad-spectrum strategy to design a group of molecular rotors, that is, a series of nonfluorescent 2-(4-vinylbenzylidene)malononitrile derivatives (BMN-Fluors), which constantly display molecular rotation in a free state. Interestingly, after activating the relevant specific-recognition reaction (i.e., hydrolysis reaction of benzylidenemalononitrile) only in the AP-site cavity within a short time (approximately 300 s), each of these molecules can be fixed into this cavity and can sequentially self-regulate to form different stable conformations in accordance with the cavity size. The different stable conformations possess various HOMO-LUMO energy gaps in their excited state. This condition enables the AP site to emit different fluorescence signals at various wavelengths. Given the different self-regulation abilities of the conformations, the series of molecules, BMN-Fluors, can emit different types of signals, including an "OFF-ON" single-channel signal, a "ratio" double-channel signal, and even a precise multichannel signal. Among the BMN-Fluors derivatives, d1-BMN can sequentially self-regulate to form five stable conformations, thereby resulting in the emission of a five-channel signal for different AP sites in situ. Thus, d1-BMN can be used as a probe to ultrasensitively recognize the AP site with precise fluorescent signals at the single-cell level. This design strategy can be generalized to develop additional single-channel to multichannel signal probes to recognize other specific sites in DNA sequences in living organisms.

A functional endonuclease Q exists in the bacterial domain: identification and characterization of endonuclease Q from Bacillus pumilus.

DNA base deamination occurs spontaneously under physiological conditions and is promoted by high temperature. Therefore, hyperthermophiles are expected to have efficient repair systems of the deaminated bases in their genomes. Endonuclease Q (EndoQ) was originally identified from the hyperthermophlic archaeon, Pyrococcus furiosus, as a hypoxanthine-specific endonuclease recently. Further biochemical analyses revealed that EndoQ also recognizes uracil, xanthine, and the AP site in DNA, and is probably involved in a specific repair process for damaged bases. Initial phylogenetic analysis showed that an EndoQ homolog is found only in the Thermococcales and some of the methanogens in Archaea, and is not present in most members of the domains Bacteria and Eukarya. A better understanding of the distribution of the EndoQ-mediated repair system is, therefore, of evolutionary interest. We showed here that an EndoQ-like polypeptide from Bacillus pumilus, belonging to the bacterial domain, is functional and has similar properties with the archaeal EndoQs.

A chemical toxicity sensor based on the electrochemiluminescence quantification of apurinic/apyrimidinic sites in double-stranded DNA monolayer

A new chemical toxicity sensor was developed based on the electrochemiluminescence (ECL) quantification of apurinic/apyrimidinic sites (AP sites) in a DNA monolayer with a covalent aldehyde reactive probe (ARP). In the sensor, a uracil-containing DNA duplex was first immobilized on a gold electrode by self-assembly. The duplex was then reacted with uracil-DNA glycosylase (UDG) to convert uracils into AP sites. ARP was employed to tag the AP site with a biotin. After reacting with a ruthenium complex labeled streptavidin, ECL was measured for quantitative analysis. The DNA monolayer was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and chronocoulometry, and its density was measured to be 2.89×10−12mol/cm2. Characterization of the reaction product between ARP and DNA AP sites in solution by nondenaturing polyacrylamide gel electrophoresis and mass spectrometry confirmed successful biotinylation. ECL intensity of the labeled DNA monolayer on the electrode was found to correlate with the number of AP sites, and the detection limit was estimated to be about 1 lesion in 512 DNA bases, which meant that 8.5 fmol AP bases on the electrode were detected. ECL response of the DNA monolayers containing either 8-oxodGuo or methylated bases was very low, indicating that ARP-based AP sites detection method was highly selective. The sensor successfully detected the AP sites in normal DNA induced by methylmethane sulfonate, a carcinogenic chemical. The novel combination of covalent probe and ECL measurement in a sensor configuration therefore provides unique advantages in selectivity and sensitivity, and can be potentially employed in the screening of chemicals for their genetic toxicity.

The induction of ligninolytic enzymes depending on menadione based oxidative stress and cell damages in Phanerochaete chrysosporium

The effect of menadione treatment on the activities of lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase, quinone detoxifying enzyme DT-diaphorase and the levels of hydroxyl (OH), total reactive oxygen species (ROS), and also protein and DNA damage in Phanerochaete chrysosporium. The activities of LiP, MnP and laccase were increased as 5.14-, 10.73- folds at 6th h and 4.18-fold at 8th h after 0.75 mM menadione treatment, respectively, compared to the controls. The highest DT-diapharose activity was found in 0.2 mM exposure as 2.48-fold of the control at 6th h. The OH and total ROS levels were higher than the controls in general. The highest OH and total ROS levels were observed in 0.5 mM treatment as 2.3-fold at 3rd h and1.9-fold at 4th h of the controls, respectively. The levels of protein carbonyl and apurinic/apyrimidinic sites in DNA increased with increasing concentration and incubation time and reached to 2.84 and 3.34-folds of their controls, respectively. In spite of a significant increase of ligninolytic enzyme activities and of DT-diaphorase activity, the increases in OH and total ROS levels caused increases in the protein carbonyl levels and AP sites in DNA by approximately 3.0-fold in P. chrysosporium. On the other hand the observed inductions in ligninolytic enzymes were higher than the increases in damage of cellular biomolecules and these results provide opportunity to develop further biotechnological applications of P. chrysosporium treated with menadione.

Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3′-end-blocking modifications, possesses DNA and RNA 3′-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3′-end of DNA have been used to prevent 3′-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3′-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3′-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3′-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.

Tyrosyl-DNA Phosphodiesterase 1 Is a New Player in Repair of Apurinic/Apyrimidinic Sites

Genomic DNA is constantly damaged by the action of exogenous factors and endogenous reactive metabolites. Apurinic/apyrimidinic sites (AP sites), which occur as a result of DNA glycosylase induced or spontaneous hydrolysis of the N-glycosidic bonds, are the most common damages of DNA. The chemical reactivity of AP sites is the cause of DNA breaks, and DNA-protein and DNA-DNA crosslinks. Repair of AP sites is one of the most important mechanisms for maintaining genome stability. Despite the fact that the main participants of the AP site repair are very well studied, the new proteins that could be involved potentially in this process as "back up" players or perform certain specialized functions are being found. This review is dedicated to one of these proteins, tyrosyl-DNA phosphodiesterase 1 (Tdp1), for which we have recently shown that in addition to its main activity of specific cleavage of the tyrosyl-DNA bond formed via a covalent attachment of topoisomerase 1 (Top1) to DNA, Tdp1 is able to initiate the cleavage of the internal AP sites in DNA and their following repair. Tdp1 was discovered in Saccharomyces cerevisiae yeast as an enzyme hydrolyzing the covalent bond between tyrosyl residue of topoisomerase 1 and 3'-phosphate group in DNA. Tdp1 is the major enzyme which carries out the repair of the irreversible complexes of DNA and topoisomerase 1, which appear. in the presence of Top 1 inhibitors, such as camptothecin, therefore Tdp1 is a very important target for the development of inhibitors--anticancer drugs. Besides, Tdp1 hydrolyzes a wide range of 3'-terminal DNA modifications and the 3'-end nucleosides and its derivatives to form a 3'-phosphate. Tdp1 ability to cleave AP sites suggests its involvement in the base excision repair as an alternative enzyme to cleave AP sites instead of AP endonuclease 1--the major enzyme hydrolyzing AP sites in DNA repair process.

Human Apurinic/Apyrimidinic Endonuclease 1

Human apurinic/apyrimidinic endonuclease 1 (APE1, also known as REF-1) was isolated based on its ability to cleave at AP sites in DNA or activate the DNA binding activity of certain transcription factors. We review herein topics related to this multi-functional DNA repair and stress-response protein. APE1 displays homology to Escherichia coli exonuclease III and is a member of the divalent metal-dependent α/β fold-containing phosphoesterase superfamily of enzymes. APE1 has acquired distinct active site and loop elements that dictate substrate selectivity, and a unique N-terminus which at minimum imparts nuclear targeting and interaction specificity. Additional activities ascribed to APE1 include 3'-5' exonuclease, 3'-repair diesterase, nucleotide incision repair, damaged or site-specific RNA cleavage, and multiple transcription regulatory roles. APE1 is essential for mouse embryogenesis and contributes to cell viability in a genetic background-dependent manner. Haploinsufficient APE1(+/-) mice exhibit reduced survival, increased cancer formation, and cellular/tissue hyper-sensitivity to oxidative stress, supporting the notion that impaired APE1 function associates with disease susceptibility. Although abnormal APE1 expression/localization has been seen in cancer and neuropathologies, and impaired-function variants have been described, a causal link between an APE1 defect and human disease remains elusive. Ongoing efforts aim at delineating the biological role(s) of the different APE1 activities, as well as the regulatory mechanisms for its intra-cellular distribution and participation in diverse molecular pathways. The determination of whether APE1 defects contribute to human disease, particularly pathologies that involve oxidative stress, and whether APE1 small-molecule regulators have clinical utility, is central to future investigations.

Mitigation of gamma-radiation induced abasic sites in genomic DNA by dietary nicotinamide supplementation: Metabolic up-regulation of NAD+ biosynthesis

The search for non-toxic radio-protective drugs has yielded many potential agents but most of these compounds have certain amount of toxicity. The objective of the present study was to investigate dietary nicotinamide enrichment dependent adaptive response to potential cytotoxic effect of 60Co γ-radiation. To elucidate the possible underlying mechanism(s), male Swiss mice were maintained on control diet (CD) and nicotinamide supplemented diet (NSD). After 6 weeks of CD and NSD dietary regimen, we exposed the animals to γ-radiation (2, 4 and 6Gy) and investigated the profile of downstream metabolites and activities of enzymes involved in NAD+ biosynthesis. Increased activities of nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase (NMNAT) were observed up to 48h post-irradiation in NSD fed irradiated mice. Concomitant with increase in liver NAMPT and NMNAT activities, NAD+ levels were replenished in NSD fed and irradiated animals. However, NAMPT and NMNAT-mediated NAD+ biosynthesis and ATP levels were severely compromised in liver of CD fed irradiated mice. Another major finding of these studies revealed that under γ-radiation stress, dietary nicotinamide supplementation might induce higher and long-lasting poly(ADP)-ribose polymerase 1 (PARP1) and poly(ADP-ribose) glycohydrolase (PARG) activities in NSD fed animals compared to CD fed animals. To investigate liver DNA damage, number of apurinic/apyrimidinic sites (AP sites) and level of 8-hydroxy-2′-deoxyguanosine (8-oxo-dG) residues were quantified. A significant increase in liver DNA AP sites and 8-oxo-dG levels with concomitant increase in caspase-3 was observed in CD fed and irradiated animals compared to NSD fed and irradiated mice. In conclusion present studies show that under γ-radiation stress conditions, dietary nicotinamide supplementation restores DNA excision repair activity via prolonged activation of PARP1 and PARG activities. Present results clearly indicated that hepatic NAD+ replenishment might be a novel and potent approach to reduce radiation injury.

Abasic site-switched structure conversion of neutral red for selective DNA recognition

DNA-binding-switched conversion of small molecule's structure and property provides a promising tool for achieving a high selectivity in identification of a DNA specific site. Herein, the optical properties of neutral red in response to its abasic site (AP site) binding were investigated for this purpose. At pH 8.4, neutral red exists in solution mainly at the neutral form (NR). However, the presence of an AP site in DNA converts NR to a protonated form (NRH+), following a 100-nm red shift in absorption spectra. This conversion is inefficient for the fully matched DNAs without the AP site. Relative to the absorption spectra, the fluorescence in response to the binding-switched conversion is more dependent on the sequences near the AP site. The AP site's hydrophobic microenvironment and electron transfer between the AP site-bound NRH+ in excited state and the nearby context bases govern the emission behavior. It was found that the AP site having a large void space favors occurrence of dipole interaction between the bound NRH+ and the context bases. This AP site-dependent conversion of neutral red in structure and then its optical properties would find wide applications in the field of DNA assay with a high selectivity and sensitivity.

Detection and Application of DNA Abasic Sites

Abasic sites (apurinic/apyrimidinic or AP sites), a common lesion in DNA, are resulted from the cleavage of n-glycosylic bond, leaving deoxyribose residue in DNA. AP sites have emerged as the mutation/cancer markers and attracted great attention. A variety of analytical methods have been developed for the determination and quantification of AP sites in DNA, including P-32 post. labeling, LC-MS, and ELISA. In this article, we provided an overview on the recent development of the DNA AP site detection using synthetic chemical probes. Covalent chemical probes were designed and synthesized which showed high specificity toward a particular AP site structure. Signal report groups were directly attached to the probe either, or indirectly by affinity reactions. Alternatively, non. covalent probes with signal groups rely on the weak (hydrogen. bonding, electrostatic, base stacking) interactions with the AP site and the consequent signal modulation. In addition, the AP sites have been explored as a recognition element in the detection of small molecules and SNPs.

MD simulation and experimental evidence for Mg²+ binding at the B site in human AP endonuclease 1.

Apurinic/apyrimidinic endonuclease 1 (APE1), a central enzyme in the base excision repair pathway, cleaves damaged DNA in Mg(2+) dependent reaction. Despite characterization of nine X-ray crystallographic structures of human APE1, in some cases, bound to various metal ions and substrate/product, the position of the metal ion and its stoichiometry for the cleavage reaction are still being debated. While a mutation of the active site E96Q was proposed to eliminate Mg(2+) binding at the "A" site, we show experimentally that this mutant still requires Mg(2+) at concentration similar to that for the wild type enzyme to cleave the AP site in DNA. Molecular dynamics simulations of the wild type APE1, E96Q and a double missense mutant E96Q + D210N indicate that Mg(2+) placed at the A-site destabilizes the bound AP site-containing DNA. In these simulations, the H-bond chain D238-H309-AP site oxygen is broken and the substrate DNA is shifted away from its crystal structure position (1DE9). In contrast, simulations with the Mg(2+) at site B or A+B sites leave the substrate DNA at the position shown in the crystal structure (1DE9). Taken together our MD simulations and biochemical analysis suggests that Mg(2+) binding at the B site is involved in the reaction mechanism associated with endonuclease function of APE1.

Oxidative DNA damage induced by iron chloride in the larvae of the lace coral Pocillopora damicornis

Biochemical and molecular biomarkers tools are utilized as early warning signatures of contaminant exposure to target and non-target organisms. The objective of this study was to investigate the sublethal effects of iron chloride to the larvae of the lace coral Pocillopora damicornis by measuring a suit of oxidative-stress biomarkers. The larvae were exposed to a range of sublethal concentrations of iron chloride (0.01, 0.1, 1, 10, and 100ppm) for seven days. With reference to oxidative stress biomarkers, the no-observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) of iron chloride were observed to be 0.01 and 100ppm respectively. At the end of the seventh day the antioxidant status of the larvae was evaluated by the levels of glutathione (GSH), glutathione peroxidase (GPX), glutathione reductase (GR), and glutathione-S-transferase (GST), in both experimental and control groups. For the quantification of cellular oxidative damage, lipid peroxidation (LPO) activity was determined in the same and the extent of DNA damage was assessed by the expression of DNA apurinic/apyrimidinic (AP) sites. Iron chloride exhibited a concentration-dependent inhibition of GSH and GPX and induction of GR, GST, LPO, and DNA-AP sites in the P. damicornis larvae when compared to the control group. The oxidative stress biomarkers of the larvae exposed to 0.1, 1, and 10ppm of iron chloride did not show any significant overall differences when compared to the control group. However the activities of LPO, GSH, GPX, GR, GST and DNA-AP in the larval group exposed to 100ppm of iron chloride exhibited statistically significant (P=0.002, 0.003, 0.002, 0.002, 0.005 and 0.007) differences when compared to the control group. The research results indicated that iron chloride in concentrations at the 100ppm level caused oxidative stress in the P. damicornis larvae.

DNA-AP sites generation by etoposide in whole blood cells.

BackgroundEtoposide is currently one of the most commonly used antitumor drugs. The mechanisms of action proposed for its antitumor activity are based mainly on its interaction with topoisomerase II. Etoposide effects in transformed cells have been described previously. The aim of the present study was to evaluate the genotoxic effects of this drug in non-transformed whole blood cells, such as occurs as collateral damage induced by some chemotherapies.MethodsTo determine etoposide genotoxicity, we employed Comet assay in two alkaline versions. To evaluate single strand breaks and delay repair sites we use pH 12.3 conditions and pH >13 to evidence alkali labile sites. With the purpose to quantified apurinic or apyrimidine (AP) sites we employed a specific restriction enzyme. Etoposide effects were determined on whole blood cells cultured in absence or presence of phytohemagglutinin (PHA) treated during 2 and 24 hours of cultured.ResultsAlkaline (pH > 13) single cell gel electrophoresis (SCGE) assay experiments revealed etoposide-induced increases in DNA damage in phytohemaglutinine (PHA)-stimulated blood and non-stimulated blood cells. When the assay was performed at a less alkaline pH, 12.3, we observed DNA damage in PHA-stimulated blood cells consistent with the existence of alkali labile sites (ALSs). In an effort to elucidate the molecular events underlying this result, we applied exonuclease III (Exo III) in conjunction with a SCGE assay, enabling detection of DNA-AP sites along the genome. More DNA AP-sites were revealed by Exo III and ALSs were recognized by the SCGE assay only in the non-stimulated blood cells treated with etoposide.ConclusionOur results indicate that etoposide induces DNA damage specifically at DNA-AP sites in quiescent blood cells. This effect could be involved in the development of secondary malignancies associated with etoposide chemotherapy.