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.     

Nucleotide excision repair of 2-acetylaminofluorene- and 2-aminofluorene-(C8)-guanine adducts: molecular dynamics simulations elucidate how lesion structure and base sequence context impact repair efficiencies

Nucleotide excision repair (NER) efficiencies of DNA lesions can vary by orders of magnitude, for reasons that remain unclear. An example is the pair of N-(2′-deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(2′-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) adducts that differ by a single acetyl group. The NER efficiencies in human HeLa cell extracts of these lesions are significantly different when placed at G₁, G₂ or G₃ in the duplex sequence (5′-CTCG₁G₂CG₃CCATC-3′) containing the NarI mutational hot spot. Furthermore, the dG-C8-AAF adduct is a better substrate of NER than dG-C8-AF in all three NarI sequence contexts. The conformations of each of these adducts were investigated by Molecular dynamics (MD) simulation methods. In the base-displaced conformational family, the greater repair susceptibility of dG-C8-AAF in all sequences stems from steric hindrance effects of the acetyl group which significantly diminish the adduct-base stabilizing van der Waals stacking interactions relative to the dG-C8-AF case. Base sequence context effects for each adduct are caused by differences in helix untwisting and minor groove opening that are derived from the differences in stacking patterns. Overall, the greater NER efficiencies are correlated with greater extents of base sequence-dependent local untwisting and minor groove opening together with weaker stacking interactions.     

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.     

The Effect of Methylation of the 6 Oxygen of Guanine on the Structure and Stability of Double Helical DNA

Abstract

The effect of methylation of the 0–6 position of guanine in short segments of double helical DNA has been investigated by molecular mechanical simulations on the sequences d(CGCGCG)₂, d(CGC+AFs-OMG+AF0-CG)₂, d(CGT+AFs-OMG+AF0-CG)₂, d(CGC+AFs-OMC+AF0-CG/(CGCGCG), d(CGC+AFs-OMG+AF0-CG/d(CGTGCG), d(CGCGAATTCGCG)₂ and d(CGCGAATTC+AFs-OMG+AF0-CG)₂. Guanines methylated at the 0–6 position are found to form hydrogen bonds of roughly equal strength to cytosine and thymine. The optimum structure of these modified base pairs are not dramatically different from normal GC pairs, but both involve some bifurcation of the proton donors of cytosine (4NH₂) or thymine (3NH) between the guanine N3 and O6 groups.     

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).     

Differential effects of N-acetyl-2-aminofluorene and N-2-aminofluorene adducts on the conformational change in the structure of DNA polymerase I (Klenow fragment)

The carcinogen N-acetyl-2-aminofluorene forms two major DNA adducts: the N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene adduct (dG-C8-AAF) and its deacetylated derivative, the N-(2'-deoxyguanosin-8-yl)-2-aminofluorene adduct (dG-C8-AF). It is well established that the AAF adduct is a very strong block for DNA synthesis in vitro while the AF adduct is more easily bypassed. In an effort to understand the molecular mechanism of this phenomenon, the structure of the complex of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) bound to primer-templates containing either an AF or AAF adduct in or near the active site was probed by nuclease and protease digestion analyses. The results of these experiments suggest that positioning the AAF adduct in the polymerase active site strongly inhibits the conformational change that is required for the insertion of a nucleotide. Similar experiments with AF-modified primer-templates shows a much less pronounced effect. The inhibition of the conformational change by either adduct is not detected if they are positioned in the single-stranded part of the template just one nucleotide before the active site. These findings may explain the different abilities of these lesions to block DNA synthesis.     

Extracellular metabolites of 2-aminofluorene in cultures of rapid and slow acetylator rabbit hepatocytes as a model for urinary and biliary metabolism

N-acetylation is involved in determining species susceptibility to carcinogenicity by certain aromatic amines. In order to further investigate this relationship, the biotransformation of 2-aminofluorene (2-AF) by monolayer cultures of hepatocytes isolated from rapid and slow acetylator rabbits was studied. Analysis of biotransformation products liberated by cells was used as an indication of metabolites that would be excreted in the urine and bile. Hepatocytes from both acetylator phenotypes were found to extensively biotransform 2-AF. The overall rates of metabolism and the types of products formed were similar in the two phenotypes, although the quantity of several products differed. Hepatocyte cultures from rapid acetylators released a greater proportion of acetylated metabolites. Rapid acetylator hepatocytes released predominantly ring-hydroxylated-2-acetylaminofluorene (2-AAF) while the major product from the slow acetylator cultures was conjugated 2-AF. The amounts of extracellular N-hydroxy-2-acetylaminofluorene were similar in both phenotypes. No phenotype-dependent differences in extracellular metabolites were noted when hepatocytes were incubated with 2-AAF. These results indicate that hepatocytes from both phenotypes have similar capacities to excrete N-hydroxy-2-AAF and to detoxify the parent aromatic amine. These findings can be related to the carcinogenicity of 2-AF in either phenotype.     

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.     

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.     

Leukemia Fusion Target AF9 Is an Intrinsically Disordered Transcriptional Regulator that Recruits Multiple Partners via Coupled Folding and Binding

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Highlights? The AF9 AHD is intrinsically disordered ? The AHD recruits AF4, BCoR, Dot1L, and hPC3 by coupled folding and binding ? AF9 binding partners compete for binding to a common site ? Dynamics of the AF4-AF9 complex may facilitate exchange between partners     

Species differences in the biochemical properties of liver microsomal arylamine and arylamide N-hydroxylases

Cytochrome P-448 dependent microsomal N-hydroxylases are key enzymes in the metabolic activation of both arylamides and arylamines.Using 2-acetylaminofluorene (2-AAF) and 2-aminofluorene (2-AF) as substrates, the present report compares the biochemical properties of rat, hamster and mouse liver N-hydroxylases. There are marked species differences both in terms of the affinity for the two substrates and in terms of maximum velocity of the enzymes. The rat and hamster liver arylamide N-hydroxylases are induced by pretreatment with 2-AAF which also significantly increases their affinity for the substrate. In mouse liver neither arylamide nor arylamine N-hydroxylases are modified or induced. With 2-AF as substrate, arylamide treatment never enhances N-hydroxylation but it reduces the K_m-value of the rat and hamster liver enzymes.Among the effectors tested in vitro, 3-methylcholanthrene (3-MC), 7,8-benzoflavone (BF), benzo[α]pyrene (B[α]P) and miconazole (MN) inhibit hepatic arylamide N-hydroxylase in the submicromolar range. Harman (H) and paraoxon (PX) act in a dose-dependent manner in the micromolar range and metyrapone (MP) is not an inhibitor even at 50-μM concentration.Among the position isomers, 1- and 3-AAF are inhibitors of the N-hydroxylating enzymes whereas 4-AAF is not.These data are discussed in relationship to the toxic effects (mutagenicity and hepatocarcinogenicity) of arylamides and arylamines with respect to the role and the complexity of their microsomal metabolism.     

Dynamics and Stability of GCAA Tetraloops with 2-Aminopurine and Purine Substitutions

Abstract

The contributions of various interactions in the GGCGCAAGCC hairpin containing a GCAA tetraloop were studied by computer simulations using the substitutions of functional groups. The guanosine (G) in the first tetraloop position or in the C-G closing base pair was replaced by 2-aminopurine (AP), and the individual tetraloop's adenosines (A) were replaced by purine (PUR). These substitutions eliminated particular hydrogen bonds thought to stabilize the GCAA tetraloop. For each substitution, molecular dynamics (MD) simulations were carried out in an aqueous solution with sodium counterions, using the CHARMM27 force field. The MD simulations showed that the substitutions in the first (G→AP) and the third (A→PUR) position of the GCAA tetraloop did not significantly influence the conformation of the hairpin. A long-lived bridging water molecule observed in the GCAA loop was present in both modified loops. The substitutions made in the last loop position (A→PUR) or in the C-G base pair closing the tetraloop (G→AP) to some extent influenced the loop structure and dynamics. These loops did not display the long- lived bridging water molecules. When the second A in the GCAA loop was replaced by PUR, the first A in the loop was observed in the anti or in the syn orientation about the gly- cosyl bond. The G to AP substitution in C-G base pair led to a change of their arrangement from the Watson-Crick to wobble. The MD simulations of the hairpin with C-AP wobble closing base pair showed increased conformational dynamics of the hairpin. The changes of hairpin formation free energy associated with the substitutions of individual bases were calculated by the free energy perturbation method. Our theoretical estimates suggest a larger destabilization for the G to AP substitutions in GCAA loop than for the substitutions of individual A's by PUR, which is in accordance with experimental tendency. The calculations predicted a similar free energy change for G to AP substitutions in the GCAA tetraloop and in the C-G closing base pair.     

Recognition and incision of site-specifically modified C8 guanine adducts formed by 2-aminofluorene, N-acetyl-2-aminofluorene and 1-nitropyrene by UvrABC nuclease

Nucleotide excision repair plays a crucial role in removing many types of DNA adducts formed by UV light and chemical carcinogens. We have examined the interactions of Escherichia coli UvrABC nuclease proteins with three site-specific C8 guanine adducts formed by the carcinogens 2-aminofluorene (AF), N-acetyl-2-acetylaminofluorene (AAF) and 1-nitropyrene (1-NP) in a 50mer oligonucleotide. Similar to the AF and AAF adducts, the 1-NP-induced DNA adduct contains an aminopyrene (AP) moiety covalently linked to the C8 position of guanine. The dissociation constants for UvrA binding to AF–, AAF– and AP–DNA adducts, determined by gel mobility shift assay, are 33 ± 9, 8 ± 2 and 23 ± 9 nM, respectively, indicating that the AAF adduct is recognized much more efficiently than the other two. Incision by UvrABC nuclease showed that AAF–DNA was cleaved ~2-fold more efficiently than AF– or AP–DNA (AAF > AF ≈ AP), even though AP has the largest molecular size in this group. However, an opened DNA structure of six bases around the adduct increased the incision efficiency for AF–DNA (but not for AP–DNA), making it equivalent to that for AAF–DNA. These results are consistent with a model in which DNA damage recognition by the E.coli nucleotide excision repair system consists of two sequential steps. It includes recognition of helical distortion in duplex DNA followed by recognition of the type of nucleotide chemical modification in a single-stranded region. The difference in incision efficiency between AF– and AAF–DNA adducts in normal DNA sequence, therefore, is a consequence of their difference in inducing structural distortions in DNA. The results of this study are discussed in the light of NMR solution structures of these DNA adducts.     

Z Helix-coil Transition of d(C-Br8G-C-G-C-Br8G) Studied by CD, 1H-NMR and IR Spectroscopies

Abstract

The conformation of díC-Bi⁸G-C-G-C-Br⁸G) in aqueous solution was studied by CD and ¹H-NMR spectroscopy and in condensed phase by IR spectroscopy. Whether in 0.1 M or 3 M NaCl solution or in film the only double helical structure adopted by brominated d(C-G)₃ oligomer is the Z form. The IR spectrum of the film presents all the characteristic absorptions of the Z conformation and in particular is indicative of a syn conformation for the central guanosine as well as for the brominated one. Imino proton resonances of diC-Bi⁸G-C- G-C-Br⁸G) demonstrating the duplex formation were observed up to 60°C. It is interesting to note that the significant highfield shifts of the dC H₅″ exocyclic sugar protons characteristic of the non exchangeable proton spectra of d(C-G)₃ containing 5-methyl dC residues in the Z form were also detected in the proton spectrum of brominated oligomer. Whereas formation of the Z helix of methylated d(C-G)₃ oligomers dependent on the salt concentration was found to occur via the preliminary formation of a B helix even in 4 M NaCl solution, the Z helix of d(C-Br⁸G-C-G-C-Br⁸G) is obtained directly from the coil form. However, IR data suggest that in the Z form of dlC-Bi⁸G-C-G-C-Bi⁸G), the overlapping of the base planes should be slightly different in comparison with the stacking observed in d(C-G)₃ crystals. The kinetic data (activation energy and lifetime) of the Z helix-coil transition of brominated d(C-G)₃ are compared to those of the B helix-coil transition observed for methylated d(C-G)₃ in 0.1 M NaCl solution while the thermodynamic data of these two reactions (enthalpy and midpoint temperature) are slightly different.     

Binding of ethidium ion to left-handed Z-RNA induces a cooperative transition to right-handed RNA at the intercalation site

The equilibrium binding of the ethidium cation (Etd+) to the right-handed A-form of poly-[r(C-G)], the B-form of poly[d(C-G)], and the left-handed Z-forms of Br-poly[r(C-G)] and Br-poly[d(C-G)] was investigated in 0.22 M NaCl by optical methods. Scatchard analysis indicates that Etd+ intercalates into right-handed forms of poly[r(C-G)] and poly[d(C-G)] in a noncooperative manner. Correlation of Etd+ absorbance binding isotherms and polynucleotide circular dichroism data indicates that drug binding to Br-poly[r(C-G) and Br-poly[d(C-G)] results in cooperative conversion from left-handed Z-forms to right-handed intercalated conformations. Approximate stoichiometries necessary to induce the left- to right-handed transitions are 1 Etd+/9 base pairs (bp) for Z-RNA and 1 Etd+/6 bp for Z-DNA. The apparent limiting binding stoichiometries are approximately 1 Etd+/3 bp for RNA and 1 Etd+/2 bp for DNA. The equilibrium binding constants for binding to the right-handed forms decrease in the order Br-poly[d(C-G)], Br-poly[r(C-G)], poly[d(C-G)], and poly[r(C-G)]. Thermodynamic parameters are obtained by van't Hoff analysis of Etd+ absorbance thermal dissociation data. Enthalpy values for all four polynucleotides are negative and of similar magnitude. Negative entropy values indicate that the binding processes are primarily enthalpically driven.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triple-helix formation in the antiparallel binding motif of oligodeoxynucleotides containing N(9)- and N(7)-2-aminopurine deoxynucleosides

Triplex-forming oligodeoxynucleotide 15mers, designed to bind in the antiparallel triple-helical binding motif, containing single substitutions (Z) of the four isomeric αN⁷-, βN⁷-, αN⁹- and βN⁹-2-aminopurine (ap)-deoxyribonucleosides were prepared. Their association with double-stranded DNA targets containing all four natural base pairs (X-Y) opposite the aminopurine residues was determined by quantitative DNase I footprint titration in the absence of monovalent metal cations. The corresponding association constants were found to be in a rather narrow range between 1.0 × 10⁶ and 1.3 × 10⁸ M^–1. The following relative order in Z × X-Y base-triple stabilities was found: Z = αN⁷ap: T-A > A-T> C-G ~ G-C; Z = βN⁷ap: A-T > C-G > G-C > T-A; Z = αN⁹ap: A-T = G-C > T-A > C-G; and Z = βN⁹ap: G-C > A-T > C-G > T-A.     

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.     

Sex differences in the biotransformation of 2-acetylaminofluorene in cultured rat hepatocytes

Sex-related differences in susceptability to 2-acetylaminofluorene (2-AAF) hepatocarcinogenicity and in vivo biotransformation of 2-AAF have been observed. In order to determine the contribution of hepatocytes to these differences, 2-AAF biotransformation was investigated in monolayer cultures of hepatocytes freshly isolated from male and female F-344 rats.In cultured hepatocytes from both sexes, ring and N-hydroxylated, deacetylated and conjugated metabolites were formed. The half-life of 2-AAF was similar at concentrations of 5×10^–6 and 10^–5 M; however, at 10^–4 M a slower rate was observed in cultures from males. Although the total formation of aqueous metabolites was similar, the ratio of sulfate to glucuronide conjugates of 2-AAF formed by hepatocytes from male and female rats differed. Sulfate conjugates predominated in hepatocytes from male rats, whereas in females, glucuronides predominated. The demonstration of sex-dependent variations in the rate of metabolism at a high concentration of 2-AAF and in conjugation provides evidence that in vivo differences are a function, at least in part, of the biotransformation characteristics of hepatocytes.Abbreviations 2-AAF 2-acetylaminofluorene - 2AF 2-aminofluorene - WME Williams Medium E.     

The Crystal Structure of d(CCCCGGGG): A New A-Form Variant with an Extended Backbone Conformation

Abstract

The crystal structure of d(CCCCGGGG) has been determined at a resolution of 2.25Å. The oligomers crystallize as A-DNA duplexes occupying crystallographic two-fold axes. The backbone conformation is, in general, similar to that observed in previously reported crystal structures of A-DNA fragments, except for the central linkage, where it adopts an extended structure resulting from all trans conformation at the P-05′-C5′-C4′ bonds. This type of conformation facilitates interstrand stacking between the guanines at the C-G site. The local helix twist at this step is very small (25°) compared to an overall average of 33.5°. The unique structure of the C-G base-pair step, namely the extended backbone and the distinct stacking geometry, may be an important feature in the recognition mechanism between double- stranded DNA molecules and restriction endonucleases such as Msp I, which cuts the sequence CCGG very specifically with a rate unaffected by neighboring base pairs.