Biomarkers of oxidative stress and DNA damage in agricultural workers: a pilot study.

Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects such as cancer and neurological diseases. A study of pesticide applicators and farmworkers was conducted to examine the relationship between organophosphate pesticide exposure and biomarkers of oxidative stress and DNA damage. Urine samples were analyzed for OP metabolites and 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Lymphocytes were analyzed for oxidative DNA repair activity and DNA damage (Comet assay), and serum was analyzed for lipid peroxides (i.e., malondialdehyde, MDA). Cellular damage in agricultural workers was validated using lymphocyte cell cultures. Urinary OP metabolites were significantly higher in farmworkers and applicators (p<0.001) when compared to controls. 8-OH-dG levels were 8.5 times and 2.3 times higher in farmworkers or applicators (respectively) than in controls. Serum MDA levels were 4.9 times and 24 times higher in farmworkers or applicators (respectively) than in controls. DNA damage (Comet assay) and oxidative DNA repair were significantly greater in lymphocytes from applicators and farmworkers when compared with controls. Markers of oxidative stress (i.e., increased reactive oxygen species and reduced glutathione levels) and DNA damage were also observed in lymphocyte cell cultures treated with an OP. The findings from these in vivo and in vitro studies indicate that organophosphate pesticides induce oxidative stress and DNA damage in agricultural workers. These biomarkers may be useful for increasing our understanding of the link between pesticides and a number of health effects.

Mechanism of action of Escherichia coli formamidopyrimidine N-glycosylase: role of K155 in substrate binding and product release.

Escherichia coli formamidopyrimidine N-glycosylase (fpg) is a DNA glycosylase with an associated beta,delta-lyase activity. We have recently shown that the highly conserved lysine residue K155 is important for base recognition. Incubation of a double-stranded DNA containing an abasic site with the wild-type fpg protein generated only beta,delta-product. However, incubation of a double-stranded DNA containing an abasic site opposite a small gap with fpg protein generated predominantly beta-product. These data suggested that the induction of a double-strand break by fpg led to the destabilization of the protein-DNA covalent intermediate, causing the fpg protein to prematurely dissociate from the DNA substrate. Furthermore, when a double-stranded DNA containing an abasic site opposite an A was used as a substrate, K155A mutant fpg protein yielded a mixture of beta- and beta,delta-products. These data suggested that K155 is essential for maintaining the stability of the intermediary protein-DNA covalent complex. Pre-steady-state burst kinetics showed that mutation in K155 led to the apparent disappearance of the initial burst, suggesting that the rate of product release from K155A is much greater than the rate of chemical reaction catalyzed by the mutant enzyme. This is consistent with the idea that K155A dissociates prematurely from the covalent complex, leading to a higher turnover number observed for K155A for DNA substrate containing an AP site.

A possible role of Ku in mediating sequential repair of closely opposed lesions.

One of the hallmarks of ionizing radiation exposure is the formation of clustered damage that includes closely opposed lesions. We show that the Ku70/80 complex (Ku) has a role in the repair of closely opposed lesions in DNA. DNA containing a dihydrouracil (DHU) close to an opposing single strand break was used as a model substrate. It was found that Ku has no effect on the enzymatic activity of human endonuclease III when the substrate DNA contains only DHU. However, with DNA containing a DHU that is closely opposed to a single strand break, Ku inhibited the nicking activity of human endonuclease III as well as the amount of free double strand breaks induced by the enzyme. The inhibition on the formation of a free double strand break by Ku was found to be much greater than the inhibition of human endonuclease III-nicking activity by Ku. Furthermore, there was a concomitant increase in the formation of Ku-DNA complexes when endonuclease III was present. Similar results were also observed with Escherichia coli endonuclease III. These results suggest that Ku reduces the formation of endonuclease III-induced free double strand breaks by sequestering the double strand breaks formed as a Ku-DNA complex. In doing so, Ku helps to avoid the formation of the intermediary free double strand breaks, possibly helping to reduce the mutagenic event that might result from the misjoining of frank double strand breaks.

Enzymatic processing of DNA containing tandem dihydrouracil by endonucleases III and VIII.

Endonuclease III from Escherichia coli, yeast (yNtg1p and yNtg2p) and human and E.coli endonuclease VIII have a wide substrate specificity, and recognize oxidation products of both thymine and cytosine. DNA containing single dihydrouracil (DHU) and tandem DHU lesions were used as substrates for these repair enzymes. It was found that yNtg1p prefers DHU/G and exhibits much weaker enzymatic activity towards DNA containing a DHU/A pair. However, yNtg2p, E. coli and human endonuclease III and E.coli endonuclease VIII activities were much less sensitive to the base opposite the lesion. Although these enzymes efficiently recognize single DHU lesions, they have limited capacity for completely removing this damaged base when DHU is present on duplex DNA as a tandem pair. Both E.coli endonuclease III and yeast yNtg1p are able to remove only one DHU in DNA containing tandem lesions, leaving behind a single DHU at either the 3'- or 5'-terminus of the cleaved fragment. On the other hand, yeast yNtg2p can remove DHU remaining on the 5'-terminus of the 3' cleaved fragment, but is unable to remove DHU remaining on the 3'-terminus of the cleaved 5' fragment. In contrast, both human endonuclease III and E.coli endonuclease VIII can remove DHU remaining on the 3'-terminus of a cleaved 5' fragment, but are unable to remove DHU remaining on the 5'-terminus of a cleaved 3' fragment. Tandem lesions are known to be generated by ionizing radiation and agents that generate reactive oxygen species. The fact that these repair glycosylases have only a limited ability to remove the DHU remaining at the terminus suggests that participation of other repair enzymes is required for the complete removal of tandem lesions before repair synthesis can be efficiently performed by DNA polymerase.

A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V.

Deoxyadenosine undergoes spontaneous deamination to deoxyinosine in DNA. Based on amino acids sequence homology, putative homologs of endonuclease V were identified in several organisms including archaebacteria, eubacteria as well as eukaryotes. The translated amino acid sequence of the Archaeoglobus fulgidus nfi gene shows 39% identity and 55% similarity to the E. coli nfi gene. A. fulgidus endonuclease V was cloned and expressed in E. coli as a C-terminal hexa-histidine fusion protein. The C-terminal fusion protein was purified to apparent homogeneity by a combination of Ni(++) affinity and MonoS cation exchange liquid chromatography. The purified C-terminal fusion protein has a molecular weight of about 25kDa and showed endonuclease activity towards DNA containing deoxyinosine. A. fulgidus endonuclease V has an absolute requirement for Mg(2+) and an optimum reaction temperature at 85 degrees C. However, in contrast to E. coli endonuclease V, which has a wide substrate spectrum, endonuclease V from A. fulgidus recognized only deoxyinosine. These data suggest that the deoxyinosine cleavage activity is a primordial activity of endonuclease V and that multiple enzymatic activities of E. coli endonuclease V were acquired later during evolution.

Detection of abasic sites and oxidative DNA base damage using an ELISA-like assay.

Reactive oxygen species produce a wide spectrum of DNA damage, including oxidative base damage and abasic (AP) sites. Many procedures are available for the quantification and detection of base damage and AP sites. However, either these procedures are laborious or the starting materials are difficult to obtain. A biotinylated aldehyde-specific reagent, ARP, has been shown to react specifically with the aldehyde group present in AP sites, resulting in biotin-tagged AP sites in DNA. The biotin-tagged AP sites can then be determined colorimetrically with an ELISA-like assay, using avidin/biotin-conjugated horseradish peroxidase as the indicator enzyme. The ARP assay is thus a simple, rapid, and sensitive method for the detection of AP sites in DNA. Furthermore, removal of damaged base by DNA N-glycosylases generates AP sites that can be measured by the ARP reagent. By coupling the ARP assay with either endonuclease III from Escherichia coli or 8-oxoguanine N-glycosylase (OGG1) from yeast, investigators can rapidly determine the amount of oxidative pyrimidine damage (endonuclease III-sensitive sites) or purine damage (OGG1-sensitive sites) in cellular DNA, respectively. An increased level of oxidative damage has been implicated in several age-related human diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease, as well as the aging process. The sensitivity and simplicity of the ARP assay thus make it a valuable method for investigators who are interested in estimating the level of oxidative DNA damage in cells and tissues derived from patients with various age-related diseases or cancers.

Characterization of a novel 8-oxoguanine-DNA glycosylase activity in Escherichia coli and identification of the enzyme as endonuclease VIII.

8-Oxoguanine (G*), induced by reactive oxygen species, is mutagenic because it mispairs with A. The major G*-DNA glycosylase (OGG), namely, OGG1 in eukaryotes, or MutM in Escherichia coli, excises G* when paired in DNA with C, G, and T, but not A, presumably because removal of G* from a G*.A pair would be mutagenic. However, repair of G* will prevent mutation when it is incorporated in the nascent strand opposite A. This could be carried out by a second OGG, OGG2, identified in yeast and human cells. We have characterized a new OGG activity in E. coli and then identified it to be endonuclease VIII (Nei), discovered as a damaged pyrimidine-specific DNA glycosylase. Nei shares sequence homology and reaction mechanism with MutM and is similar to human OGG2 in being able to excise G* when paired with A (or G). Kinetic analysis of wild type Nei showed that it has significant activity for excising G* relative to dihydrouracil. The presence of OGG2 type enzyme in both E. coli and eukaryotes, which is at least as efficient in excising G* from a G*.A (or G) pair as from a G*.C pair, supports the possibility of G* repair in the nascent DNA strand.

Deoxyxanthosine in DNA is repaired by Escherichia coli endonuclease V.

Deoxycytidine, deoxyadenosine and deoxyguanosine undergo spontaneous deamination to form deoxyuridine, deoxyinosine and deoxyxanthosine, respectively. In this manuscript, we show that in addition to its known ability to recognize deoxyuridine and deoxyinosine in DNA, Escherichia coli endonuclease V cleaves DNA containing deoxyxanthosine. However, Alk A protein and human methylpurine glycosylase are unable to recognize deoxyxanthosine. Endonuclease V cleaves DNA containing deoxyxanthosine at the second phosphodiester bond 3' to deoxyxanthosine, generating a 3'-hydroxyl and a 5'-phosphoryl group at the nick site. This endonucleolytic activity requires Mg(2+) or Mn(2+), and is highly specific for double stranded DNA. Endonuclease V-catalyzed cleavage of DNA containing deoxyxanthosine is a result of its ability to recognize the altered base and not due to its mismatch-specific endonuclease activity. The ability of endonuclease V to recognize both deoxyinosine and deoxyxanthosine suggests that endonuclease V is important for preventing mutations that might arise as a result of deamination of purines.

Mutation identification DNA analysis system (MIDAS) for detection of known mutations.

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA detection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/ apurinic (AP) site is cleaved by AP endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32p- or FITC-labeled oligonucleotides as mutation probes. Generally between 20-50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full-length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser-induced fluorescence-capillary electrophoresis (LIF-CE) of fluorophore-labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3'-endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG-MRE). MIDAS differentiated between a clinical sample BRCA 1 wild-type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF-CE should make detection of known point mutations, deletions, and insertions a rapid and cost-effective technique well suited for automation.

Yield of DNA strand breaks after base oxidation of plasmid DNA.

We have irradiated aerobic aqueous solutions of plasmid DNA with 137Cs gamma rays in the presence of inorganic radical scavengers including nitrite, iodide, azide, thiocyanate and bromide. These scavengers react with the strongly oxidizing hydroxyl radical (*OH) to produce less powerful oxidants. Of these scavengers, only thiocyanate and bromide result in the formation of oxidizing species [(SCN)2*- and Br2*-, respectively] which are capable of reacting with the bases in DNA. The oxidized bases were detected after incubation of the irradiated plasmid with the two E. coli DNA base excision repair endonucleases, formamidopyrimidine-DNA N-glycosylase and endonuclease III. Depending on the experimental conditions, the intermediate base radicals may ultimately form stable oxidized bases in very high yields (within an order of magnitude of the *OH yield), and possibly also single-strand breaks (SSBs) in much lower yield (between 0.1 and 1% of the total yield of base damage). By competing for (SCN)2*- with an additional species (nitrite), it was possible to estimate the second-order rate constant for the reaction of (SCN)2*- with DNA as 1.6 x 10(4) dm3 mol(-1) s(-1), and also to demonstrate a correlation between the large yield of damaged bases and the much smaller increase in the yield of SSBs over background levels due to *OH. The efficiency of transfer of damage from oxidized base to sugar is estimated as about 0.5% or 5%, depending on whether purine or pyrimidine base radicals are responsible for the base to sugar damage transfer.

Uracil glycol deoxynucleoside triphosphate is a better substrate for DNA polymerase I Klenow fragment than thymine glycol deoxynucleoside triphosphate.

A major stable oxidation product of DNA cytosine is 5,6-dihydroxy-5, 6-dihydrouracil (Ug). Ug can be formed directly in DNA or in the cellular nucleotide pools by deamination of the unstable primary product, cytosine glycol. Here, we synthesized dUgTP and showed that dUgTP was incorporated in place of dTTP and was a much better substrate for the model enzyme DNA polymerase I Klenow fragment lacking proofreading activity, Kf (exo-), than deoxythymidine glycol triphosphate (dTgTP). The relative efficiency for dUgTP insertion opposite A was 10 times higher than for dTgTP; however, the extension of a primer with 3' dUg was about 100 times more efficient than the extension of a primer with 3' dTg. At the insertion step, the differences in Vmax appeared to be responsible since the apparent Kms for dUgTP and dTgTP were about the same. In contrast, both the apparent Km and Vmax for elongation of dUg were markedly different from those of dTg. Molecular modeling was performed with both Tg and Ug and provides a rational structural explanation for these observations.

Asbestos increases mammalian AP-endonuclease gene expression, protein levels, and enzyme activity in mesothelial cells.

Only two DNA repair enzymes, DNA polymerase beta and O6-methylguanine-DNA methyltransferase, have been shown to be inducible in mammalian cells by genotoxic agents. We show here that crocidolite asbestos induces the DNA repair enzyme, apurinic/apyrimidinic (AP)-endonuclease, in isolated mesothelial cells, the progenitor cells of malignant mesothelioma. Asbestos at nontoxic concentrations of 1.25 and 2.5 microg/cm2 significantly increased AP-endonuclease mRNA and protein levels as well as enzyme activity (P < 0.05) in a dose-dependent manner in rat pleural mesothelial cells. These increases were persistent from 24 to 72 h after initial exposure to fibers. Changes were not observed with glass beads, a noncarcinogenic particle. Confocal scanning laser microscopy showed that AP-endonuclease was primarily localized in the nucleus but also in mitochondria. Our data are the first to demonstrate the inducibility of AP-endonuclease by a human class I carcinogen associated with oxidant stress in normal cells of the lung.

Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions.

One of the most prevalent lesions in DNA is the apurinic/apyrimidinic (AP) site, which is derived from the cleavage of the N-glycosyl bond by DNA glycosylase or by spontaneous depurination. AP sites are repaired by AP endonucleases during the process of base excision repair; however, an imbalance in this DNA repair system may cause mutations as well as cell death. We have established a sensitive and convenient slot-blot method to detect AP sites in genomic DNA using a novel aldehyde reactive probe (ARP), which reacts with the aldehydic group of ring-opened AP sites. The reaction of 1 mM of ARP with 15 microg of genomic DNA containing AP sites at 37 degrees C was completed within 1 min. The AP site-ARP complex was remarkably stable during incubation in TE buffer, even at 100 degrees C for 60 min. The sensitivity of this assay enables detection of 2.4 AP sites per 10(7) bases. By using this ARP-slot-blot assay, the rate of spontaneous depurination of calf thymus DNA was determined. Under physiological conditions, AP sites were increased at 1.54 AP sites/10(6) nucleotides/day (9000 AP sites/cell/day). This highly sensitive assay allows us to determine the endogenous level of AP sites in genomic DNA, as well as to investigate whether DNA-damaging agents cause imbalances of base excision/AP endonuclease repair in vivo and in vitro.

Antibodies to oxidative DNA damage: characterization of antibodies to 8-oxopurines.

The 8-oxo-7,8-dihydropurines (8-oxopurines) are important cellular premutagenic lesions produced in DNA by free radicals. Specific antibodies were prepared to detect these lesions. For antigens, 8-oxo-7,8-dihydroadenosine (8-oxoAdo) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) were synthesized from the bromonucleosides, and the immunogens were produced by conjugating these to either bovine serum albumin or rabbit serum albumin by the periodate method. Polyclonal antibodies specific for the haptens were elicited from rabbits immunized with the BSA conjugates. The antibodies to 8-oxoAdo (anti-8-oxoAdo) and 8-oxoGuo (anti-8-oxoGuo) precipitated the homologous antigens in an Ouchterlony gel diffusion assay and no cross-reactivity was observed toward the normal nucleosides or to the heterologous 8-oxopurine. Specificity was also examined by hapten inhibition of antibody reactivity with the homologous conjugates using ELISA. For anti-8-oxoAdo, the IC50 for 8-oxoAdo was 8 mumol/L and 8-bromoadenosine, guanosine, and inosine did not inhibit, even at concentrations of 1.25 mmol/L. Similarly, the IC50 for anti-8-oxoGuo for 8-oxoGuo was 0.1 mumol/L. 8-Methoxyguanosine also inhibited the reaction but was about 500-fold less effective than the eliciting hapten. Other nucleosides tested did not inhibit at concentrations up to 100 mumol/L. Both antibodies could easily detect the corresponding damage in x-irradiated f1 DNA at a dose of 7.5 Gy and both antibodies recognized the corresponding lesion in duplex DNA; however, with anti-8-oxoGuo the signal was reduced about 50% compared to single-stranded DNA. In order to determine the exact amount of each lesion produced in irradiated DNA, and to standardize the ELISA signal, both products were measured after alkaline phosphatase digestion of x-irradiated calf thymus DNA using high-pressure liquid chromatography (HPLC) coupled to an electrochemical detector. Anti-8-oxoGuo could detect ten 8-oxoG residues and anti-8-oxoAdo could detect two 8-oxoA residues per 10,000 nucleotides. Thus, these antibodies should be useful for the detection and measurement of 8-oxopurines in cellular DNA.

Mechanism of action of base release by Escherichia coli Fpg protein: role of lysine 155 in catalysis.

Fpg protein (formamidopyrimidine/8-oxoguanine DNA N-glycosylase) is a DNA repair enzyme that catalyzes the removal of oxidized purines, most notably the mutagenic 7-hydro-8-oxoguanine (8oxoGua) lesion, by an N-glycosylase action. Additionally, Fpg protein catalyzes beta and delta elimination reactions subsequent to removal of the base lesions, as well as the analogous chemistry at abasic sites (AP sites). In this report, we show that of the two lysines that are conserved among the various putative prokaryotic Fpg proteins, a site specific alteration in one of them (lysine 155 changed to alanine) displays meaningful changes in substrate activities. However, lysine 155 is not required for the postulated covalent enzyme-substrate imine intermediate as demonstrated by trapping of the mutant protein-oligonucleotide complexes with cyanide or cyanoborohydride. The K155A mutant shows a decrease in activity with the 8oxoGua-substrate of approximately 50-fold under both k(cat)/Km and k(cat) conditions. This mutant also displays a similar reduction in activity with an oligonucleotide substrate possessing a single 2'-deoxy-8-oxonebularine site. In contrast, activity for a site specific 7-methylformamidopyrimidine-modified oligonucleotide is reduced approximately 3-4-fold, a much more modest decrease in activity. Interestingly, there is a concomitant increase in AP lyase activity above wild-type for the K155A mutant (1.6-fold increase in k(cat), 32-fold increase in k(cat)/Km), demonstrating retention of functional beta and delta lyase activities. Together these observations are readily accommodated by a model requiring a direct interaction of lysine 155 with the C8 oxygen of 8-oxopurines. Thus, conservation of this amino acid residue during evolution appears to be essential for specific incision of the mutagenic 8oxoGua base lesion by Fpg protein.

Patterns of 8-hydroxydeoxyguanosine formation in DNA and indications of oxidative stress in rat and human pleural mesothelial cells after exposure to crocidolite asbestos.

Oxidative damage is a proposed mechanism of asbestos-induced carcinogenesis, but the detection of oxidative DNA lesions in target cells of asbestos-induced mesothelioma has not been examined. In studies here, DNA was isolated from both rat pleural mesothelial (RPM) cells and a human mesothelial cell line (MET5A) after exposure in vitro to crocidolite asbestos at various concentrations. DNA was then examined for formation of 8-hydroxydeoxyguanosine (8-OHdG) at 24, 48 and 72 h using HPLC with electrochemical detection. In addition, steady-state mRNA levels of manganese-containing superoxide dismutase (MnSOD) were assessed as an indication of oxidative stress. Whereas RPM cells showed dose-dependent and significant increases in 8-OHdG formation in response to crocidolite asbestos or iron-chelated crocidolite fibers (but not after exposure to glass beads), MET5A cells showed decreases in 8-OHdG. Both cell types exhibited elevations in message levels of MnSOD. In comparison with human MET5A cells, RPM cells exhibited increased cytotoxicity and apoptosis in response to asbestos, as documented by cell viability assays and flow cytometry analysis using propidium iodide. Results in RPM cells indicate that asbestos causes oxidative damage that may result in potentially mutagenic lesions in DNA and/or apoptosis, despite compensatory increases in expression of an antioxidant enzyme.

Characterization of Escherichia coli endonuclease VIII.

Escherichia coli endonuclease VIII (endo VIII) was identified as an enzyme that, like endonuclease III (endo III), removes radiolysis products of thymine including thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, and urea from double-stranded plasmid or phage DNA and cleaves the DNA strand at abasic (AP) sites (Melamede, R. J., Hatahet, Z., Kow, Y. W., Ide., H., and Wallace, S. S. (1994) Biochemistry 33, 1255-1264). Using apparently homogeneous endo VIII protein, we now show that endo VIII removes from double-stranded oligodeoxyribonucleotides the stable oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil. Endo VIII cleaved the damage-containing DNA strand by beta,delta-elimination as does formamidopyrimidine DNA glycosylase (Fpg). Like Fpg, endo VIII also excised the 5'-terminal deoxyribose phosphate from an endonuclease IV (endo IV) pre-incised AP site. Thus, in addition to amino acid sequence homology (Jiang, D., Hatahet, Z., Blaisdell, J., Melamede, R. J., and Wallace, S. S. (1997) J. Bacteriol. 179, 3773-3782), endo VIII shares a number of catalytic properties with Fpg. In addition, endo VIII specifically bound to oligodeoxynucleotides containing a reduced AP site with a stoichiometry of 1:1 for protein to DNA with an apparent equilibrium dissociation constant of 3.9 nM. Like Fpg and endo III, the DNase I footprint was small with contact sites primarily on the damage-containing strand; unlike Fpg and endo III, the DNA binding of endo VIII to DNA was asymmetric, 3' to the reduced AP site.

Further characterization of Escherichia coli endonuclease V. Mechanism of recognition for deoxyinosine, deoxyuridine, and base mismatches in DNA.

Endonuclease V from Escherichia coli has a wide substrate spectrum. In addition to deoxyinosine-containing DNA, the enzyme cleaves DNA containing urea residues, AP sites, base mismatches, insertion/deletion mismatches, flaps, and pseudo-Y structures. The gene coding for the enzyme was identified to be orf 225 or nfi (endonuclease five). Using enzyme purified from an overproducing strain, the deoxyinosine- and mismatch-specific activities of endonuclease V was found to have different divalent metal requirements. The affinity of the enzyme is greater than 20-fold higher for DNA containing deoxyinosine than deoxynebularine or base mismatches. Under optimal cleavage conditions, endonuclease V forms two stable complexes with DNA containing deoxyinosine, but not with DNA containing base mismatches or deoxynebularine, suggesting that the 6-keto group of hypoxanthine in DNA is critical for stable interactions with the protein. The enzyme recognizes deoxyuridine in DNA but exhibits a much lower affinity to DNA containing deoxyuridine compared with DNA containing deoxyinosine. Interestingly, deoxyuridine-specific endonuclease activity of endonuclease V has a divalent metal requirement similar to the mismatch activity. A model for the mechanism of substrate recognition is proposed to explain these different activities.

A common mechanism of action for the N-glycosylase activity of DNA N-glycosylase/AP lyases from E. coli and T4.

Duplex oligonucleotides containing the base lesion analogs, O-methylhydroxylamine- and O-benzylhydroxylamine-modified abasic (AP) sites, were substrates for the DNA N-glycosylases endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. These N-glycosylases are known to have associated AP lyase activities. In contrast, uracil DNA N-glycosylase, a simple N-glycosylase which does not have an associated AP lyase activity, was unable to recognize the modified AP sites. Endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V recognized the base lesion analogs as N-glycosylases generating intermediary AP sites which were subsequently cleaved by the enzyme-associated AP lyase activities. Kinetic measurements showed that O-alkoxyamine-modified AP sites were poorer substrates than the presumed physiological substrates. For endonuclease III, DNA containing O-methylhydroxyl-amine or O-benzylhydroxylamine was recognized at 12 and 9% of the rate of DNA containing thymine glycol, respectively, under subsaturating substrate concentrations (as determined by relative Vmax/K(m)). Similarly, with formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. DNA containing O-methylhydroxylamine or O-benzylhydroxylamine was recognized at 4-9% of the efficiency of DNA containing N7-methyl formamidopyrimidine or pyrimidine cyclobutane dimers, respectively. Based on the known structures of these base lesion analogs and the substrate specificities of the N-glycosylases, a common mechanism of action is proposed for DNA N-glycosylases with an associated AP lyase activity.