Abasic Sites In DNA Research Articles

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe.

Modulation of the current signatures of DNA abasic site adducts in the α-hemolysin ion channel

Electrical current signatures of DNA adducts were investigated during immobilization of strands inside the membrane-bound α-hemolysin ion channel. The current blockages produced by these adducts were found to depend on both size and shape, providing insights into the DNA-protein interactions and the size limitation of bulky adducts to be translocated.

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells.

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.

A Microenvironment‐Sensitive Fluorescent Pyrimidine Ribonucleoside Analogue: Synthesis, Enzymatic Incorporation, and Fluorescence Detection of a DNA Abasic Site

Base-modified fluorescent ribonucleoside-analogue probes are valuable tools in monitoring RNA structure and function because they closely resemble the structure of natural nucleobases. Especially, 2-aminopurine, a highly environment-sensitive adenosine analogue, is the most extensively utilized fluorescent nucleoside analogue. However, only a few isosteric pyrimidine ribonucleoside analogues that are suitable for probing the structure and recognition properties of RNA molecules are available. Herein, we describe the synthesis and photophysical characterization of a small series of base-modified pyrimidine ribonucleoside analogues derived from tagging indole, N-methylindole, and benzofuran onto the 5-position of uracil. One of the analogues, based on a 5-(benzofuran-2-yl)pyrimidine core, shows emission in the visible region with a reasonable quantum yield and, importantly, displays excellent solvatochromism. The corresponding triphosphate substrate is effectively incorporated into oligoribonucleotides by T7 RNA polymerase to produce fluorescent oligoribonucleotide constructs. Steady-state and time-resolved spectroscopic studies with fluorescent oligoribonucleotide constructs demonstrate that the fluorescent ribonucleoside photophysically responds to subtle changes in its environment brought about by the interaction of the chromophore with neighboring bases. In particular, the emissive ribonucleoside, if incorporated into an oligoribonucleotide, positively reports the presence of a DNA abasic site with an appreciable enhancement in fluorescence intensity. The straightforward synthesis, amicability to enzymatic incorporation, and sensitivity to changes in the microenvironment highlight the potential of the benzofuran-conjugated pyrimidine ribonucleoside as an efficient fluorescent probe to investigate nucleic acid structure, dynamics, and recognition events.

Dynamics of Uracil and 5-Fluorouracil in DNA

The prodrug 5-fluorouracil (5-FU), after activation into 5-F-dUMP, is an extensively used anticancer agent that inhibits thymidylate synthase and leads to increases in dUTP and 5-F-dUTP levels in cells. One mechanism for 5-FU action involves DNA polymerase mediated incorporation of dUTP and 5-F-dUTP into genomic DNA leading to U/A, 5-FU/A, or 5-FU/G base pairs. These uracil-containing lesions are recognized and excised by several human uracil excision repair glycosylases (hUNG2, hSMUG2, and hTDG) leading to toxic abasic sites in DNA that may precipitate cell death. Each of these enzymes uses an extrahelical base recognition mechanism, and previous studies with UNG have shown that extrahelical recognition is facilitated by destabilized base pairs possessing kinetically enhanced base pair opening rates. Thus, the dynamic properties of base pairs containing 5-FU and U are an important unknown in understanding the role of these enzymes in damage recognition and prodrug activation. The pH dependence of the (19)F NMR chemical shift of 5-FU imbedded in a model trinucleotide was used to obtain a pK(a) = 8.1 for its imino proton (10 °C). This is about 1.5 units lower than the imino protons of uracil or thymine and indicates that at neutral pH 5-FU exists significantly as an ionized tautomer that can mispair with guanine during DNA replication. NMR imino proton exchange measurements show that U/A and 5-FU/A base pairs open with rate constants (k(op)) that are 6- and 13-fold faster than a T/A base pair in the same sequence context. In contrast, these same base pairs have apparent opening equilibrium constants (αK(op)) that differ by less than a factor of 2, indicating that the closing rates (k(cl)) are enhanced by nearly equal amounts as k(op). These dynamic measurements are consistent with the previously proposed kinetic trapping model for extrahelical recognition by UNG. In this model, the enhanced intrinsic opening rates of destabilized base pairs allow the bound glycosylase to sample dynamic extrahelical excursions of thymidine and uracil bases as the first step in recognition.

Oxidative DNA damage in lung tissue from patients with COPD is clustered in functionally significant sequences

Lung tissue from COPD patients displays oxidative DNA damage. The present study determined whether oxidative DNA damage was randomly distributed or whether it was localized in specific sequences in either the nuclear or mitochondrial genomes. The DNA damage-specific histone, gamma-H2AX, was detected immunohistochemically in alveolar wall cells in lung tissue from COPD patients but not control subjects. A PCR-based method was used to search for oxidized purine base products in selected 200 bp sequences in promoters and coding regions of the VEGF, TGF-β1, HO-1, Egr1, and β-actin genes while quantitative Southern blot analysis was used to detect oxidative damage to the mitochondrial genome in lung tissue from control subjects and COPD patients. Among the nuclear genes examined, oxidative damage was detected in only 1 sequence in lung tissue from COPD patients: the hypoxic response element (HRE) of the VEGF promoter. The content of VEGF mRNA also was reduced in COPD lung tissue. Mitochondrial DNA content was unaltered in COPD lung tissue, but there was a substantial increase in mitochondrial DNA strand breaks and/or abasic sites. These findings show that oxidative DNA damage in COPD lungs is prominent in the HRE of the VEGF promoter and in the mitochondrial genome and raise the intriguing possibility that genome and sequence-specific oxidative DNA damage could contribute to transcriptional dysregulation and cell fate decisions in COPD.

Endonuclease G plays a role in immunoglobulin class switch DNA recombination by introducing double-strand breaks in switch regions

Immunoglobulin (Ig) class switch DNA recombination (CSR) is the crucial mechanism diversifying the biological effector functions of antibodies. Generation of double-strand DNA breaks (DSBs), particularly staggered DSBs, in switch (S) regions of the upstream and downstream CH genes involved in the specific recombination process is an absolute requirement for CSR. Staggered DSBs would be generated through deamination of dCs on opposite DNA strands by activation-induced cytidine deaminase (AID), subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and abasic site nicking by apurinic/apyrimidic endonuclease. However, consistent with the findings that significant amounts of DSBs can be detected in the IgH locus in the absence of AID or Ung, we have shown in human and mouse B cells that AID generates staggered DSBs not only by cleaving intact double-strand DNA, but also by processing blunt DSB ends generated in an AID-independent fashion. How these AID-independent DSBs are generated is still unclear. It is possible that S region DNA may undergo AID-independent cleavage by structure-specific nucleases, such as endonuclease G (EndoG). EndoG is an abundant nuclease in eukaryotic cells. It cleaves single and double-strand DNA, primarily at dG/dC residues, the preferential sites of DSBs in S region DNA. We show here that EndoG can localize to the nucleus of B cells undergoing CSR and binds to S region DNA, as shown by specific chromatin immunoprecipitation assays. Using knockout EndoG−/− mice and EndoG−/− B cells, we found that EndoG deficiency resulted in a two-fold reduction in CSR in vivo and in vitro, as demonstrated by reduced cell surface IgG1, IgG2a, IgG3 and IgA, reduced secreted IgG1, reduced circle Iγ1-Cμ, Iγ3-Cμ, Iɛ-Cμ, Iα-Cμ transcripts, post-recombination Iμ-Cγ1, Iμ-Cγ3, Iμ-Cɛ and Iμ-Cα transcripts. In addition to reduced CSR, EndoG−/− mice showed a significantly altered spectrum of mutations in IgH JH-iEμ DNA. Impaired CSR in EndoG−/− B cells did not stem from altered B cell proliferation or apoptosis. Rather, it was associated with significantly reduced frequency of DSBs. Thus, our findings determine a role for EndoG in the generation of S region DSBs and CSR.

Effect of 8-oxoguanine and abasic site DNA lesions on in vitro elongation by human DNA polymerase ϵ in the presence of replication protein A and proliferating-cell nuclear antigen

DNA pol (polymerase) is thought to be the leading strand replicase in eukaryotes. In the present paper, we show that human DNA pol can efficiently bypass an 8-oxo-G (7,8-dihydro-8-oxoguanine) lesion on the template strand by inserting either dCMP or dAMP opposite to it, but it cannot bypass an abasic site. During replication, DNA pols associate with accessory proteins that may alter their bypass ability. We investigated the role of the human DNA sliding clamp PCNA (proliferating-cell nuclear antigen) and of the human single-stranded DNA-binding protein RPA (replication protein A) in the modulation of the DNA synthesis and translesion capacity of DNA pol . RPA inhibited the elongation by human DNA pol on templates annealed to short primers. PCNA did not influence the elongation by DNA pol and had no effect on inhibition of elongation caused by RPA. RPA inhibition was considerably reduced when the length of the primers was increased. On templates bearing the 8-oxo-G lesion, this inhibitory effect was more pronounced on DNA replication beyond the lesion, suggesting that RPA may prevent extension by DNA pol after incorporation opposite an 8-oxo-G. Neither PCNA nor RPA had any effect on the inability of DNA pol to replicate past the AP site, independent of the primer length.

Conformational Transitions in Human AP Endonuclease 1 and Its Active Site Mutant during Abasic Site Repair

AP endonuclease 1 (APE1) is a crucial enzyme of the base excision repair pathway (BER) in human cells. APE1 recognizes apurinic/apyrimidinic (AP) sites and makes a nick in the phosphodiester backbone 5' to them. The conformational dynamics and presteady-state kinetics of wild-type APE1 and its active site mutant, Y171F-P173L-N174K, have been studied. To observe conformational transitions occurring in the APE1 molecule during the catalytic cycle, we detected intrinsic tryptophan fluorescence of the enzyme under single turnover conditions. DNA duplexes containing a natural AP site, its tetrahydrofuran analogue, or a 2'-deoxyguanosine residue in the same position were used as specific substrates or ligands. The stopped-flow experiments have revealed high flexibility of the APE1 molecule and the complexity of the catalytic process. The fluorescent traces indicate that wild-type APE1 undergoes at least four conformational transitions during the processing of abasic sites in DNA. In contrast, nonspecific interactions of APE1 with undamaged DNA can be described by a two-step kinetic scheme. Rate and equilibrium constants were extracted from the stopped-flow and fluorescence titration data for all substrates, ligands, and products. A replacement of three residues at the enzymatic active site including the replacement of tyrosine 171 with phenylalanine in the enzyme active site resulted in a 2 x 10(4)-fold decrease in the reaction rate and reduced binding affinity. Our data indicate the important role of conformational changes in APE1 for substrate recognition and catalysis.

CoQ10 conveys protection from oxidative stress in plasma but not skeletal muscle

CoQ10 conveys protection from oxidative stress in plasma but not skeletal muscle Annateresa Papazzo1, Louise Lexis2, Paul Lewandowski11Molecular Nutrition Unit, School of Medicine, Deakin University, Victoria, Australia; 2School of Human Biosciences, La Trobe University, Victoria, AustraliaAbstract: Coenzyme Q10 (CoQ10) is commonly consumed as an antiaging supplement at doses of 30–210 mg/day. The aim of the study was to determine if CoQ10 alters markers of antioxidant status, oxidative damage, and gene expression in aging skeletal muscle. Female guinea pigs aged 26 months were supplemented for 6 weeks with CoQ10 at a human equivalent dose of 10 mg/kg/day. Body weight, plasma CoQ10 concentration, and WBC DNA abasic sites were measured at weeks 0, 2, 4, and 6 of the supplementation period. At the end of supplementation, concentrations of skeletal muscle CoQ10, glutathione, malondialdehyde, protein carbonyls, DNA abasic sites, activities of catalase and glutathione peroxidase, and the gene expression of cyctochrome c oxidase subunits were measured. Dietary supplementation with CoQ10 elevated plasma CoQ10 levels (pre 73 ± 3 nmol/L, post 581 ± 15 nmol/L, P < 0.05) and decreased abasic sites in WBC DNA (pre 16.8 ± 0.5 Ap/100000 bp, post 9.7 ± 0.4 Ap/100000 bp, P < 0.05). In contrast, all of the measures made in skeletal muscle were not different between groups (P > 0.05). These results indicate that dietary supplementation with CoQ10 at a dose of 10 mg/kg/day may be capable of increasing antioxidant protection and reducing oxidative damage in the plasma, but may have no effect in skeletal muscle.Keywords: coenzyme Q10, aging, skeletal muscle, oxidative damage, gene expression

Chemical shift assignments for human apurinic/apyrimidinic endonuclease 1

Apurinic/apyrimidinic endonuclease 1 (APE1 or Ref-1) is the major enzyme in mammals for processing abasic sites in DNA. These cytotoxic and mutagenic lesions arise via spontaneous rupture of the base-sugar bond or the removal of damaged bases by a DNA glycosylase. APE1 cleaves the DNA backbone 5' to an abasic site, giving a 3'-OH primer for repair synthesis, and mediates other key repair activities. The DNA repair functions are essential for embryogenesis and cell viability. APE1-deficient cells are hypersensitive to DNA-damaging agents, and APE1 is considered an attractive target for inhibitors that could potentially enhance the efficacy of some anti-cancer agents. To enable an important new method for studying the structure, dynamics, catalytic mechanism, and inhibition of APE1, we assigned the chemical shifts (backbone and (13)C(beta)) of APE1 residues 39-318. We also report a protocol for refolding APE1, which was essential for achieving complete exchange of backbone amide sites for the perdeuterated protein.

Development of novel chemical probes to detect abasic sites in DNA

We chemically synthesized a series of aminooxy derivatives to develop novel probes for sensitive detection of abasic (AP) sites in DNA. The results of the conjugation reactions showed that the probes could efficiently react to AP sites by introducing an aromatic or a guanidino group in their structures. In particular, the probe having both functional groups showed the most effective reactivity, indicating that hydrophobic and electrostatic interactions cooperatively acted in the reaction of the probe to AP sites. We then synthesized a biotinylated probe and succeeded in more sensitive detection of AP sites in genomic DNA than with the conventional aldehyde reactive probe (ARP).

Weight Loss in Obese Men Is Associated with Increased Telomere Length and Decreased Abasic Sites in Rectal Mucosa

Telomere shortening may cause genome instability and is an initiating event in colorectal cancer (CRC). Obesity is associated with reduced telomere length in lymphocytes and is a risk factor for CRC, but the impact of obesity on telomere length in the rectal mucosa is unknown. The purpose of this study was to investigate the effect of weight loss, induced by calorie-restricted diets, on telomere length in the rectal mucosa of obese men. Midrectal biopsies were collected by sigmoidoscopy at three time points (at weeks 0, 12, and 52) during a programmed weight loss intervention. Weight was reduced by an average of 10.6 kg across the study. Telomere length, measured by quantitative real-time PCR (qPCR), was negatively correlated with body mass index (BMI) (r = -0.13, p = 0.05) at baseline (n = 54) and increased at week 12 (four-fold increase) and week 52 (10-fold increase) (analysis of covariance [ANCOVA] p = 0.01, n = 12). Abasic sites in DNA decreased at week 12 (30% decrease) and week 52 (65% decrease) (analysis of variance [ANOVA] p = 0.02). Furthermore, gain of telomere length appeared to be greater if more weight and body fat was lost (r = -0.65, p = 0.01 and r = -0.56, p = 0.01, respectively). These results suggest that weight loss by caloric-restricted diets may contribute to the prevention of telomere shortening and DNA base damage, which are important initiating events in carcinogenesis.

Escherichia coli HU protein has a role in the repair of abasic sites in DNA

HU is one of the most abundant DNA binding proteins in Escherichia coli. We find that it binds strongly to DNA containing an abasic (AP) site or tetrahydrofuran (THF) (apparent K(d) approximately 50 nM). It also possesses an AP lyase activity that cleaves at a deoxyribose but not at a THF residue. The binding and cleavage of an AP site was observed only with the HUalphabeta heterodimer. Site-specific mutations at K3 and R61 residues led to a change in substrate binding and cleavage. Both K3A(alpha)K3A(beta) and R61A(alpha)R61A(beta) mutant HU showed significant reduction in binding to DNA containing AP site; however, only R61A(alpha)R61A(beta) mutant protein exhibited significant loss in AP lyase activity. Both K3A(alpha)K3A(beta) and R61K(alpha)R61K(beta) showed slight reduction in AP lyase activities. The function of HU protein as an AP lyase was confirmed by the ability of hupA or hupB mutations to further reduce the viability of an E. coli dut(Ts) xth mutant, which generates lethal AP sites at 37 degrees C; the hupA and hupB derivatives, respectively, had a 6-fold and a 150-fold lower survival at 37 degrees C than did the parental strain. These data suggest, therefore, that HU protein plays a significant role in the repair of AP sites in E. coli.

Ochratoxin A–Mediated DNA and Protein Damage: Roles of Nitrosative and Oxidative Stresses

Ochratoxin A (OTA) is a mycotoxin occurring in a variety of foods. OTA is nephrotoxic and nephrocarcinogenic in rodents. An OTA-mediated increase of the inducible nitric oxide synthase (iNOS) expression was observed in normal rat kidney renal cell line and in rat hepatocyte cultures, suggesting the induction of nitrosative stress. This was associated with an increased nuclear factor kappa-light chain enhancer of activated B cells activity. The potential consequences of iNOS induction were further investigated. A significant increase in the levels of protein nitrotyrosine residues was observed with OTA. In addition, OTA was found to increase the level of DNA abasic sites in both cell cultures system. This end point was used as an indirect measure of 8-nitroguanine formation. Treatment of the cells with L-N(6)-(1-iminoethyl) lysine, a specific inhibitor of iNOS activity, inhibited the OTA-mediated overnitration of proteins but did not reduce the level of DNA abasic sites. It was found previously that nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) activators were able to restore the cellular defense against oxidative stress and could prevent DNA abasic sites in cell cultures. In the present study, pretreatment of the cells with activators of Nrf2 prevented OTA-mediated increase in lipid peroxidation, confirming the potential of Nrf2 activators to confer protection against OTA-mediated oxidative stress. In addition, it was found that Nrf2 activators could also prevent OTA-induced protein nitration and cytotoxicity. In conclusion, the present data further confirm oxidative stress as a key source of OTA-induced DNA damage and provide additional evidence for a role of this mechanism in OTA carcinogenicity. The exact role of nitrosative stress still remains to be established.

Enhanced conformational sampling of nucleic acids by a new Hamiltonian replica exchange molecular dynamics approach

Although molecular dynamics (MD) simulations have been applied frequently to study flexible molecules, the sampling of conformational states separated by barriers is limited due to currently possible simulation time scales. Replica-exchange (Rex)MD simulations that allow for exchanges between simulations performed at different temperatures (T-RexMD) can achieve improved conformational sampling. However, in the case of T-RexMD the computational demand grows rapidly with system size. A Hamiltonian RexMD method that specifically enhances coupled dihedral angle transitions has been developed. The method employs added biasing potentials as replica parameters that destabilize available dihedral substates and was applied to study coupled dihedral transitions in nucleic acid molecules. The biasing potentials can be either fixed at the beginning of the simulation or optimized during an equilibration phase. The method was extensively tested and compared to conventional MD simulations and T-RexMD simulations on an adenine dinucleotide system and on a DNA abasic site. The biasing potential RexMD method showed improved sampling of conformational substates compared to conventional MD simulations similar to T-RexMD simulations but at a fraction of the computational demand. It is well suited to study systematically the fine structure and dynamics of large nucleic acids under realistic conditions including explicit solvent and ions and can be easily extended to other types of molecules.

Induction of abasic sites by the drinking-water mutagen MX in Salmonella TA100

Mutagen X (MX) is a chlorinated furanone that accounts for more of the mutagenic activity of drinking water than any other disinfection by-product. It is one of the most potent base-substitution mutagens in the Salmonella (Ames) mutagenicity assay, producing primarily GC to TA mutations in TA100. MX does not produce stable DNA adducts in cellular or acellular DNA. However, theoretical calculations predict that it might induce abasic sites, which it does in supercoiled plasmid DNA but not in rodents. To investigate the ability of MX to induce abasic sites in cellular DNA, we used an aldehydic site assay to detect abasic sites in DNA from Salmonella TA100 cells treated for 1.5 h with MX. At 0, 2.3, and 4.6 μM, MX induced mutant frequencies (revertants/10 6 survivors) and percent survivals of 2 (100%), 14.9 (111%), and 59.3 (45%), respectively. The frequencies of abasic sites (sites/10 5 nucleotides) for the control and two concentrations were 5.9, 6.2, and 9.7, respectively, with the frequency at the highest concentration being significant ( P < 0.001). These results provide some evidence for the ability of MX to induce abasic sites in cellular DNA. However, the lack of a dose response makes it unclear whether this DNA damage underlies the mutagenic activity of MX.

Detection of Abasic Sites in DNA by Electrochemical, Immunoelectrochemical and Acoustic Methods Using OsO4, 2,2′‐bipyridine as a Probe for Unpaired Thymine Residues

AbstractWe report on comparative analysis of the detection of DNA damage modeled by presence of abasic sites (AP) at defined positions using the chemical modification of the DNA by the complex of osmium tetroxide‐2,2‐bipyridine (Os, bipy) that selectively binds to unpaired thymine residues in the damaged DNA. AP were detected by electrochemical detection (EC) of the Os, bipy‐thymine adducts, by immunoelectrochemical (IE) and by thickness shear mode acoustic methods (TSM). EC method of detection can perfectly distinguish between the number of AP. IE and TSM methods were of comparable sensitivity.

An AP Endonuclease 1–DNA Polymerase β Complex: Theoretical Prediction of Interacting Surfaces

Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5′ to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-β). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-β, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-β based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-β located downstream of APEX1 (3′ to the damaged site) and three with pol-β located upstream of APEX1 (5′ to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (∼−10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-β at the 3′-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-β in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-β in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3′ side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available.

Mutagenicity of 8-nitroguanosine, a product of nitrative nucleoside modification by reactive nitrogen oxides, in mammalian cells

8-Nitroguanosine is a nitratively modified nucleoside that is formed endogeneously under inflammatory conditions dependent on nitric oxide production, particularly associated with cancer risks. Here, we investigated the mutagenic potential of 8-nitroguanosine in mammalian cells. Treatment with 8-nitroguanosine (10–1000 μM) for 1 h significantly increased (by 6–8 times) the mutation frequency of the xanthine–guanine phosphoribosyltransferase ( gpt) gene in AS52 cells without cytotoxic effects. 8-Nitroguanosine treatment induced a G-to-T transversion in gpt gene at position 86. It also significantly increased levels of abasic sites in DNA. These observations suggest that formation of 8-nitroguanosine may contribute to the pathogenesis of inflammation-associated carcinogenesis.