Recognition and repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease

Recognition of damage induced by N-hydroxy-2-aminofluorene (N-OH-AF) and N-acetoxy-2-(acetylamino)fluorene (NAAAF) in both phi X174 RFI supercoiled DNA and a linear DNA fragment by purified UVRA, UVRB, and UVRC proteins was investigated. We have previously demonstrated that N-OH-AF and NAAAF treatments produce N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-C8-AAF), respectively, in DNA. Using a piperidine cleavage method and DNA sequence analysis, we have found that all guanine residues can be modified by N-OH-AF and NAAAF. These two kinds of adducts have different impacts on the DNA helix structure; while dG-C8-AF maintains the anti configuration, dG-C8-AAF is in the syn form. phi X174 RF DNA-Escherichia coli transfection results indicate that while the uvrA, uvrB, and uvrC gene products are needed to repair dG-C8-AAF, the uvrC, but not the uvrA or uvrB gene products, is needed for repair of dG-C8-AF. However, we have found that in vitro the UVRA, UVRB, and UVRC proteins must work in concert to nick both dG-C8-AF and dG-C8-AAF. In general, the reactions of UVRABC nuclease toward dG-C8-AF are similar to those toward dG-C8-AAF; it incises seven to eight nucleotides from the 5' side and three to four nucleotides from the 3' side of the DNA adduct. Evidence is presented to suggest that hydrolysis on the 3' and 5' sides of the damaged base by UVRABC nuclease is not simultaneous and that at least occasionally hydrolysis occurs only on the 3' side or on the 5' side of the damage site. The possible mechanisms of UVRABC nuclease incision for AF-DNA are discussed.     

DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coli. A correlation between the conformation of the premutagenic lesion and the mutation specificity

When the chemical carcinogen N-2-acetylaminofluorene binds to DNA in vivo, two major adducts are formed, both at position C-8 of the guanine residue. One of these (the acetylaminofluorene adduct) retains the acetyl group, while the other (the aminofluorene adduct) is the corresponding deacetylated form. Unlike -AAF adducts, which trigger important structural changes of the DNA secondary structure (either the insertion-denaturation model or the induction of a Z-DNA structure, depending upon the local nucleotide sequence), -AF adducts bind to the C-8 of guanine residues without causing any major conformational change of the B-DNA structure. Well-defined adducts (either -AF or -AAF) can be formed in vitro by reacting DNA with either N-hydroxy-N-2-aminofluorene or N-acetoxy-N-2-acetylaminofluorene. Specific cleavage of the phosphodiester backbone at -AF adducts can be achieved by treating -AF-modified DNA in 1 M-piperidine at 90 degrees C. This observation led us to construct the spectrum for -AF binding to a defined DNA restriction fragment. It is found that only guanine residues react to form alkali-labile lesions and that the reactivity among the different guanines is similar. In a forward mutation assay, namely the inactivation of the tetracycline resistance gene, we found previously that more than 90% of mutations induced by -AAF adducts are frameshift mutations. Using the same assay, we show here that -AF adducts induce primarily base substitution mutations (85%), mainly of the G to T transversion type. There is therefore a strong correlation between the nature of the carcinogen-induced conformational change of the DNA structure and the corresponding mutation specificity. The -AF-induced base substitution mutations depend upon the umuC gene function(s). The data obtained in our forward mutation assay are compared to the data previously obtained in the histidine reversion assay (Ames test).     

Mutational spectrum and recombinogenic effects induced by aminofluorene adducts in bacteriophage M13

Double-stranded replicative form (RFI) DNA of bacteriophage M13 strain M13mp10 which carries partial lacZ gene has been modified in vitro to various extents with N-hydroxy-2-amino-fluorene (N-OH-AF) and then transfected into E. coli cells. High-performance liquid chromatography (HPLC) analysis results demonstrate that the sole adduct (95%) formed in modified DNA is N-(deoxyguanosine-8-yl)-2-aminofluorene (dG-C8-AF). Approximately 20 adducts per RFI molecule constitute 1 lethal event when plaque-forming ability is assayed on E. coli cells which have received no prior SOS induction. The mutagenicity of dG-C8-AF adducts was assayed by measuring loss of beta-galactosidase activity as a function of adducts per molecule. A dose-dependent increase in Lac- mutants was observed, with a 4-fold increase in mutants per survivor at 30 adducts/molecule. The mutations produced, characterized by DNA sequencing, occur predominantly at either G or C positions different from those observed in the spontaneous mutant spectrum. Restriction-mapping results show that in our assay system, dG-C8-AF adducts induce a previously unreported recombinogenic activity.     

A circular dichroism study on the conformation of d(CGT) modified with N-acetyl-2-aminofluorene or 2-aminofluorene.

The trinucleotide d(CGT) was modified by covalent binding of the carcinogen N-acetyl-2-aminofluorene (AAF) or 2-aminofluorene (AF) at the C8 position of the guanine base. The conformations of d(CGT)-AAF and -AF were studied by comparing the absorption and circular dichroism properties with those of dCMP + dGMP-AAF or -AF + dTMP in a molar ratio of 1:1:1 and AAF- and AF-containing dGMP. For both AAF- and AF-d(CGT) complexes the results show significant stacking interactions between the fluorene residue and the base(s) and are discussed in terms of the conformation of d(CGT)-AAF and -AF. In d(CGT)-AF we observe a clear interaction between AF and thymine, whereas the C-G stack is still intact. In the case of d(CGT)-AAF the C-G stack is weakened and the glycosidic rotation angle of dGuo-C8-AAF is most probably syn. The specific fluorene-base interactions persist at elevated temperatures. The carcinogen-base interactions are stronger in the AAF-carrying d(CGT) than in the case of the deacetylated complex. This is consistent with the higher mobility of the AF-adduct and its conformationally heterogeneous appearance in DNA.     

Alternative conformations of DNA modified by N-2-acetylaminofluorene

Modification of DNA by the carcinogen N-acetoxy-N-2-acetylaminofluorene gives two adducts, a major one at the C-8 position of guanine and a minor one at the N-2 position with differing conformations. Binding at the C-8 position results in a large distortion of the DNA helix referred to as the “base displacement model” with the carcinogen inserted into the DNA helix and the guanosine displaced to the outside. The result is increased susceptibility to nuclease S, digestion due to the presence of large, single-stranded regions in the modified DNA. In contrast, the N-2 adduct results in much less distortion of the helix and is less susceptible to nuclease S₁ digestion. A third and predominant adduct is formed in vivo, the deacetylated C-8 guanine adduct. The conformation of this adduct has been investigated using the dimer dApdG as a model for DNA. The attachment of aminofluorene (AF) residues introduced smaller changes in the circular dichroism (CD) spectra of dApdG than binding of acetylaminofluorene (AAF) residues. Similarly, binding of AF residues caused lower upfield shifts for the H-2 and H-8 protons of adenine than the AAF residues. These results suggest that AF residues are less stacked with neighboring bases than AAF and induce less distortion in conformation of the modified regions than AAF. An alternative conformation of AAF-modified deoxyguanosine has been suggested based on studies of poly(dG-dC)·poly(dG-dC). Modification of this copolymer with AAF to an extent of 28% showed a CD spectrum that had the characteristics of the left-handed Z conformation seen in unmodified poly-(dG-dC)·poly(dG-dC) at high ethanol or salt concentrations. Poly(dG)·poly(dC), which docs not undergo the B to Z transition at high ethanol concentrations, did not show this type of conformational change with high AAF modification. Differences in conformation were suggested by single-strand specific nuclease S₁ digestion and reactivity with anticytidine antibodies. Highly modified poly(dG-dC)·poly(dG-dC) was almost completely resistant to nuclease S₁ hydrolysis, while, modified DNA and poly(dG)·poly(dC) are highly susceptible to digestion. Two possible conformations for deoxyguanosine modified at the C-8 position by AAF are compared depending on whether its position is in alternating purine-pyrimidine sequences or random sequence DNA.     

Formation and removal of DNA adducts in liver of rats treated with hepatocarcinogens 2-aminofluorene or 2-acetylaminofluorene.

The level of adducts in DNA of rats treated with 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) was compared at the times from 1 h till 28 days after injection. The highest amount of DNA adducts was observed 12 h after treatment with 2-AF and 24 h after treatment with 2-AAF, and reached values of about 18 and 21 fmol per micrograms DNA, respectively. Participation of the nonacetylated form, dG-C8-AF, in the total amount of DNA adducts was only slightly greater in rats treated with 2-AF then in those treated with 2-AAF.     

A Circular Dichroism Study on the Conformation of d(CGT) Modified with N-acetyl-2-aminofluorene or 2-aminofluorene

Abstract

The trinucleotide d(CGT) was modified by covalent binding of the carcinogen N-acetyl-2- aminofluorene (AAF) or 2-aminofluorene (AF) at the C8 position of the guanine base. The conformations of d(CGT)-AAF and -AF were studied by comparing the absorption and circular dichroism properties with those of dCMP + dGMP-AAF or -AF + dTMP in a molar ratio of 1:1:1 and AAF- and AF-containing dGMP. For both AAF- and AF- d(CGT) complexes the results show significant stacking interactions between the fluorene residue and the base(s) and are discussed in terms of the conformation of d(CGT)-AAF and - AF. In d(CGT)-AF we observe a clear interaction between AF and thymine, whereas the C-G stack is still intact. In the case of d(CGT)-AAF the C-G stack is weakened and the glycosidic rotation angle of dGuo-C8-AAF is most probably syn. The specific fluorene-base interactions persist at elevated temperatures. The carcinogen-base interactions are stronger in the AAF-carrying d(CGT) than in the case of the deacetylated complex. This is consistent with the higher mobility of the AF-adduct and its conformationally heterogeneous appearance in DNA.     

Quantification of aminofluorene adduct formation and repair in defined DNA sequences in mammalian cells using the UVRABC nuclease

Using the UVRABC nuclease as a reagent coupled with DNA restriction and hybridization analysis we have developed a method to quantify N-acetoxy-2-acetylaminofluorene (NAAAF)-induced DNA damage in the coding and noncoding sequences of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. High performance liquid chromatography analysis shows that the only DNA adduct formed in NAAAF-treated CHO cells is N-(deoxyguanosine-C8-yl)-2-aminofluorene (dG-C8-AF). DNA sequencing analysis demonstrates that the UVRABC nuclease incises at all potential sites in which dG-C8-AF adduct may form in linear DNA fragments. We have found that the formation and removal of dG-C8-AF adducts in the coding and 3' downstream noncoding sequences of the DHFR domain are similar in cells treated with 10 microM NAAAF (3.1 adducts/14 kilobases); DNA adduct removal attains 70% for both sequences within 24 h. This result contrasts with that obtained for the repair of cyclobutane dipyrimidines in the DHFR gene, in which the repair efficiency is much higher in the coding region than in the 3' downstream noncoding region. Our results suggest that in CHO cells the repair pathway for aminofluorene DNA adducts is not the same as that for cyclobutane dipyrimidines. This new technique has the potential to detect a variety of chemical carcinogen induced DNA adducts at the gene level in cultured cells and in DNA isolated from animal tissues.     

Interaction of fluorescently labeled myosin subfragment 1 with nucleotides and actin

Isolated myosin heads (subfragment 1) were modified by covalent attachment of 5-(iodoacetamido)fluorescein or 5-(iodoacetamido)salicylic acid to the essential sulfhydryl group SH1. The extrinsic fluorescence of the modified proteins was sensitive to binding of nucleotides and F-actin. With the fluorescein derivative [subfragment 1 (S1) modified with 5-(iodoacetamido)fluorescein (IAF) at SH1 (S1-AF)], association with MgADP decreased the probe fluorescence by 30%, whereas binding to actin increased the emission by a factor of 2. In the ternary complex acto-S1-AF X MgADP, the effect of nucleotide on the intensity of the attached fluorescein canceled the effect of actin. The fluorescence state of this ternary complex was similar to that of S1-AF X MgADP. The emission of S1-AF was resolved into two components with lifetimes of 4.3 and 0.6 ns and relative contributions of 33% and 67%, respectively. Interaction of S1-AF with nucleotides and actin did not alter the lifetimes but significantly shifted their fractional contributions. Quenching studies showed that the short lifetime likely arose from the fluorescein moiety statically quenched by internal groups. Binding of MgADP to the salicylate derivative [S1 modified with 5-(iodoacetamido)salicylic acid at SH1 (S1-SAL)] induced a 25% enhancement of the probe fluorescence, whereas formation of acto-S1-SAL decreased the emission by 10% regardless of whether MgADP was bound to the protein. Both labeled S1 species bound MgADP with a similar affinity, comparable to that of unmodified S1 previously reported by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a one-electron mechanism of 2-aminofluorene oxidation by prostaglandin H synthase and horseradish peroxidase

Previous studies have shown that the primary arylamine carcinogen 2-aminofluorene (2-AF) is oxidized by the prostaglandin H synthase peroxidase to mutagenic and electrophilic products capable of covalent binding to macromolecules. The present study was designed to identify the potential reactive intermediate(s) responsible for binding, and to characterize further the metabolic intermediates in 2-AF peroxidation. Both prostaglandin H synthase and horseradish peroxidase, with H2O2, oxidize 2-AF to azofluorene, 2-aminodifluorenylamine (2-ADFA), 2-nitrofluorene, polymeric and nonorganic-extractable material. Both enzymes show greater activity at pH 5.0 than at pH 7.0. In the presence of either 2-t-butyl-4-methoxyphenol or 2,6-dimethylphenol, arylamine/phenol adducts were formed in high yield, with the nitrogen of either 2-AF or 2-ADFA coupled to the para position of the phenol (loss of -OCH3 with 2-t-butyl-4-methoxyphenol). These structures were confirmed by mass spectrometry and NMR spectroscopy. Acid hydrolysis of N-hydroxy-2-AF to yield the nitrenium ion, in the presence of a phenol, also results in adduct formation, but only at times greater than 2 h and in very limited yield. The peroxidase-catalyzed adduct formation, however is rapid (less than 2 min) and extensive. These and other data support a one-electron pathway for 2-AF peroxidation, with a free radical or a free radical-derived product responsible for binding to protein and DNA. An N-hydroxy intermediate may therefore not be obligatory in the enzymatic activation of 2-AF to a mutagenic product.     

Crystal structure of NADP(H)-dependent 1,5-anhydro-D-fructose reductase from Sinorhizobium morelense at 2.2 A resolution: construction of a NADH-accepting mutant and its application in rare sugar synthesis

Recombinant 1,5-anhydro-d-fructose reductase (AFR) from Sinorhizobium morelense S-30.7.5 was crystallized in complex with the cofactor NADP(H) and its structure determined to 2.2 A resolution using selenomethionine SAD (refined R(work) and R(free) factors of 18.9 and 25.0%, respectively). As predicted from the sequence and shown by the structure, AFR can be assigned to the GFO/IDH/MocA protein family. AFR consists of two domains. The N-terminal domain displays a Rossmann fold and contains the cofactor binding site. The intact crystals contain the oxidized cofactor NADP(+), whose attachment to the cofactor binding site is similar to that of NADP(+) in glucose-fructose oxidoreductase (GFOR) from Zymomonas mobilis. Due to variations in length and sequence within loop regions L3 and L5, respectively, the adenine moiety of NADP(+) adopts a different orientation in AFR caused by residue Arg38 forming hydrogen bonds with the 2'-phosphate moiety of NADP(+) and cation-pi stacking interactions with the adenine ring. Amino acid replacements in AFR (S10G, A13G, and S33D) showed that Ala13 is crucial for the discrimination between NADPH and NADH and yielded the A13G variant with dual cosubstrate specificity. The C-terminal domain contains the putative substrate binding site that was occupied by an acetate ion. As determined by analogy to GFOR and by site-directed mutagenesis of K94G, D176A, and H180A, residues Lys94, Asp176, and His180 are most likely involved in substrate binding and catalysis, as substitution of any of these residues resulted in a significant decrease in k(cat) for 1,5-AF. In this context, His180 might serve as a general acid-base catalyst by polarizing the carbonyl function of 1,5-AF to enable the transfer of the hydride from NADPH to the substrate. Here we present the first structure of an AFR enzyme catalyzing the stereoselective reduction of 1,5-AF to 1,5-anhydro-d-mannitol, the final step of a modified anhydrofructose pathway in S. morelense S-30.7.5. We also emphasize the importance of the A13G variant in biocatalysis for the synthesis of 1,5-AM and related derivatives.     

The Escherichia coli O6-methylguanine-DNA methyltransferase does not repair promutagenic O6-methylguanine residues when present in Z-DNA

The repair of O6-methylguanine present in N-methylnitrosourea (MNU)-treated alternating polynucleotides MNU-poly(dG-dC) X poly(dG-dC) and MNU-poly(dG-me5dC) X poly(dG-me5dC] was investigated using O6-methylguanine-DNA methyltransferase purified from Escherichia coli. Both modified polynucleotides are equally good substrates for the DNA methyltransferase when they are in the B-form. The substrate properties of the MNU-treated polynucleotides do not differ from those of MNU-treated DNA. One of these modified polynucleotides, MNU-poly(dG-me5dC) X (dG-me5dC), can adopt the Z-conformation under physiological conditions. The conformational transition of the poly(dG-me5dC) X poly(dG-me5dC) from the B-form to the Z-form was monitored by the modification of its spectroscopic properties and by the specific binding of antibodies raised against Z-DNA. The O6-methylguanine residues are repaired in MNU-poly(dG-me5dC) X poly(dG-me5dC) in B-form. At variance, the conversion of this template to the Z-form completely inhibits the repair of the O6-methylguanine residues. The cooperative transition from the Z- to the B-form of MNU-poly(dG-me5dC) X poly(dG-me5dC), mediated by intercalating drugs such as ethidium bromide, restores the ability of MNU-poly(dG-me5dC) X poly(dG-me5dC) to be substrate for the transferase. These results imply that the promutagenic DNA lesion O6-methylguanine persists in Z-DNA fragments and suggest that DNA conformation modulates the extent of DNA repair and, as a result, plays an important role in determining the mutagenic potency of chemical carcinogens.     

Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N(2)()-yl)-2-acetylaminofluorene (dG-N(2)-AAF) DNA adducts. The dG-N(2)-AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha were strongly blocked at a position one base before dG-N(2)-AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing (5)(')TCCTCCTCXCCTCTC (X = dG-N(2)-AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N(2)-AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol eta, but not pol kappa, may play a role in this process.     

Live cell imaging of outward and inward vesiculation induced by the complement c5b-9 complex

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.     

Spectrophotometrical and immunochemical studies on the conformational changes in poly(dG-dC).poly(dG-dC) after modification by 4-hydroxyaminoquinoline 1-oxide

Poly(dG-dC).poly(dG-dC) was modified by the reaction with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP. The conformations of 4HAQO-modified poly(dG-dC).poly(dG-dC) and of poly(dG-dC).poly(dG-dC) were studied by circular dichroism spectra under various salt concentration conditions. 4HAQO residues to guanine bases are inefficient in inducing the transition of poly(dG-dC).poly(dG-dC) from B-form to Z-form conformation. We have elicited monoclonal antibodies against 4HAQO-poly(dG-dC).poly(dG-dC). They were characterized using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and binding to supercoiled DNA. These antibodies reacted with 4HAQO-poly(dG-dC).poly(dG-dC) specifically but not with 4HAQO-modified DNA or poly(dG).poly(dC). However, they cross-reacted with N-acetoxy-2-acetylaminofluorene-modified poly(dG-dC).poly(dG-dC) in Z-form conformation. These monoclonal antibodies may recognize a unique conformation in poly(dG-dC).poly(dG-dC) after 4HAQO modification.     

Effect of chemical modifications on the K99 and K88ab fibrillar adhesins of Escherichia coli

The role of specific amino acid residues of the K88ab and K99 fibrillar adhesins in the binding to erythrocytes and antibodies has been studied by chemical modification. It appeared that: (1) The integrity of the single disulfide bridge in the K99 subunits is essential for the binding of the fibrillae to the glycolipid receptors, but not for the recognition and binding of specific anti-K99 antibodies. (2) Modification of one lysine residue per subunit with 4-chloro-3,5-dinitrobenzoate results in the loss of the adhesive capacity of K99 fibrillae. Lysine residue are not important for the adhesive activity of K88ab fibrillae. Three or five lysine residues per subunit, respectively, can be modified without an effect on the immunological properties of the K99 and K88ab fibrillae. (3) Limited reaction of K99 and K88ab fibrillae with 2,3-butanedione destroys the adhesive activity of both fibrillae. This inactivation corresponds with the loss of one (K99) or two (K88ab) arginine residues per subunit. Ultimately, in K99 three, and in K88ab four, arginine residues per subunit can be modified without affecting the binding of specific antibodies. (4) Modification of five out of the nine carboxyl groups contained in the K99 subunit suppresses the recognition of specific anti-K99 antibodies, but carboxylates are not important for the adhesive activity of K99 fibrillae. Modification of two additional carboxylates in K99 results in an insoluble product. (5) Tyrosine residues are most probably not present in the adhesive or antigenic sites of K99 fibrillae. Modification of six out of the ten tyrosine residues in the K88ab subunit results in a decrease in adhesive activity but has no effect on the reaction with anti-K88ab antibodies.     

Immunological detection of lesions in DNA

The purpose of this paper is to show that the antibodies to nucleic acids, to nucleosides or to DNA damaged by a physical or a chemical agent, are useful tools in the study of DNA damage and repair. The results obtained with antibodies to nucleosides, antibodies to nucleosides and DNA modified by chemical carcinogens emphasize the potential of immunological methods in three main areas, a) the sensitive detection and quantitation of adducts; b) the visualization of adducts in tissues, individual cells, and along the DNA double helix; c) the study of conformational changes of DNA induced by adducts.     

Mutagenic response of mouse lymphoma cells after activation of benzidine and 2-aminofluorene with purified prostaglandin H synthase

The mutagenic response of L5178Y mouse lymphoma cells to the model aromatic amine carcinogens, benzidine (BNZ) and 2-aminofluorene (2-AF) in the presence of the mammalian peroxidase prostaglandin H synthase (PHS) was examined. Standard incubation conditions for mouse lymphoma cells and the PHS system were developed. The cells were exposed to BNZ and 2-AF with purified PHS in the presence or absence of a peroxide, 5-phenyl-4-pentenyl hydroperoxide (PPHP) which is required for PHS-dependent amine oxidation. Incubations were carried out in a medium consisting of Hanks' balanced salt solution with calcium and magnesium and 0.1% pluronic F-68. BNZ by itself or in the presence of PPHP induced a weak mutagenic response in mouse lymphoma cells, but the addition of PHS or PHS and its co-factor PPHP increased the mutagenic response approximately 5-fold over that observed in the absence of PHS. A maximal mutagenic response for BNZ was observed after incubation with the complete activating system, PHS and PPHP. These data are in agreement with the fact that BNZ is an excellent substrate for PHS. When 2-AF was incubated with mouse lymphoma cells, only a minimal mutagenic response was observed. Incubation of 2-AF with either PPHP or PPHP and PHS (complete peroxidase system produced a significant enhancement in mutagenic response. Thus, the mutagenic response of the mouse lymphoma cells to 2-AF was dependent on the peroxide, PPHP but not the enzyme PHS. These data suggest that 2-AF, which is a poor PHS substrate, is oxidized by a different catalyst than PHS. This work demonstrates that BNZ and 2-AF are converted by peroxide-dependent mechanisms to mutagens that can be detected in mammalian cells.     

Induction of sister-chromatid exchanges by 2-aminofluorene in cultured human lymphocytes with and without erythrocytes.

2-Aminofluorene (2-AF), an indirect mutagen reported to be metabolically activated by erythrocytes in the Salmonella mutagenicity test, was studied for the induction of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro with (whole-blood cultures) and without erythrocytes (isolated lymphocyte cultures). 2-AF (0.025-0.8 mM) was present in the cultures for the last 48 h of 72-h cultures. In both types of culture, SCEs increased in a dose-dependent manner, with a statistically significant elevation already at the lowest concentration of 2-AF tested and maximum responses of 2.4-fold (whole blood) and 2.1-fold (isolated lymphocytes), in comparison with mean SCEs/cell in control cultures, at 0.4 and 0.2 mM concentrations (respectively). Thus, the induction of SCEs by 2-AF was not dependent on the presence of erythrocytes. Styrene (2 mM), a positive control chemical known to require erythrocytes for efficient SCE induction in vitro, was shown to produce a 4.9-fold increase in SCEs in whole-blood cultures, but only a slight (1.3-fold) effect in isolated lymphocyte cultures. The results suggest that leukocytes, but not erythrocytes, are important in the metabolic activation of 2-AF in the human lymphocyte SCE assay.     

Immunochemical detection of 7-methylguanine residues in nucleic acids

The ability of specific antibodies to react with 7-methylguanine residues in nucleic acids was investigated. Anti-7-methylguanine specific antibodies precipitated polymers of poly-guanylic acid which were methylated to an extent of 35 or 70% at the N-7 position of guanine, indicating that these antibodies could readily detect 7-methylguanine residues in a polynucleotide. This reaction was proportional to the total amount of 7-methylguanine present, suggesting further that quantitation of these residues is possible. To determine the minimal amount required for detection, varying amounts of 7-methylguanine were introduced into calf thymus DNA by alkylation with dimethyl sulfate. While showing no reaction with denatured nonalkylated DNA, the reaction of antibodies with alkylated DNA was proportional to the amount of 7-methylguanine in the preparations. Moreover, the antibodies appeared to detect differences in the distribution of 7-methylguanine residues in extensively methylated DNA. Precipitation was observed with DNA containing as little as one 7-methylguanine residue per 300 nucleotides, suggesting that these antibodies can be used to detect biologically significant levels of 7-methylguanine in viral and cellular nucleic acids.