Mapping the binding site of aflatoxin B1 in DNA: systematic analysis of the reactivity of aflatoxin B1 with guanines in different DNA sequences

The mutagenic and carcinogenic chemical aflatoxin B1 (AFB1) reacts almost exclusively at the N(7)-position of guanine following activation to its reactive form, the 8,9-epoxide (AFB1 oxide). In general N(7)-guanine adducts yield DNA strand breaks when heated in base, a property that serves as the basis for the Maxam-Gilbert DNA sequencing reaction specific for guanine. Using DNA sequencing methods, other workers have shown that AFB1 oxide gives strand breaks at positions of guanines; however, the guanine bands varied in intensity. This phenomenon has been used to infer that AFB1 oxide prefers to react with guanines in some sequence contexts more than in others and has been referred to as "sequence specificity of binding". Herein, data on the reaction of AFB1 oxide with several synthetic DNA polymers with different sequences are presented, and (following hydrolysis) adduct levels are determined by high-pressure liquid chromatography. These results reveal that for AFB1 oxide (1) the N(7)-guanine adduct is the major adduct found in all of the DNA polymers, (2) adduct levels vary in different sequences, and, thus, sequence specificity is also observed by this more direct method, and (3) the intensity of bands in DNA sequencing gels is likely to reflect adduct levels formed at the N(7)-position of guanine. Knowing this, a reinvestigation of the reactivity of guanines in different DNA sequences using DNA sequencing methods was undertaken. The reactivities of 190 guanines were determined quantitatively and considered in a pentanucleotide context, 5'-WXGYZ-3', where the central, underlined G represents the reactive guanine and W, X, Y, and Z can be any of the nucleotide bases. Methods are developed to determine that the X (5'-side) base and the Y (3'-side) base are most influential in determining guanine reactivity. The influence of the bases in the 5'-position (X) is 5'-G (1.0) greater than C (0.8) greater than A (0.3) greater than T (0.2), while the influence of the bases in the 3'-position (Y) is 3'-G (1.0) greater than T (0.8) greater than C (0.4) greater than A (0.3). These rules in conjunction with molecular modeling studies (to be published elsewhere) were used to assess the binding sites that might be utilized by AFB1 oxide in its reaction with DNA.     

A viral genome containing an unstable aflatoxin B1-N7-guanine DNA adduct situated at a unique site.

A problem that has hindered the study of the biological properties of certain DNA adducts, such as those that form at the N7 atoms of purines, is their extreme chemical lability. Conditions are described for the construction of a single-stranded genome containing the chemically and thermally labile 8,9-dihydro-8- (N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct, the major DNA adduct of the potent liver carcinogen aflatoxin B1 (AFB1). A 13mer oligonucleotide, d(CCTCTTCGAACTC), was allowed to react with the exo-8,9-epoxide of AFB1 to form an oligonucleotide containing a single AFB1-N7-Gua (at the underlined guanine). This modified 13mer was 5'-phosphorylated and ligated into a gap in an M13 bacteriophage genome generated by annealing a 53mer uracil-containing scaffold to M13mp7L2 linearized by EcoRI. Following ligation, the scaffold was enzymatically removed with uracil DNA glycosylase and exonuclease III. The entire genome construction was complete within 3 h and was carried out at 16 degrees C, pH 6.6, conditions determined to be optimal for AFB1-N7-Gua stability. Characterization procedures indicated that the AFB1-N7-Gua genome was approximately 95% pure with a small (5%) contamination by unmodified genome. This construction scheme should be applicable to other chemically or thermally unstable DNA adducts.     

The structure of the aflatoxin B1-DNA adduct at N7 of guanine. Theoretical intercalation and covalent adduct models

Two theoretical models are proposed for the conformational structure of both intercalated and covalent adduct complexes of aflatoxin B1, designated AFB1, with N7 of guanine of DNA. The covalent adduct model requires the DNA to kink a minimum of 39 degrees about the covalent site of the C8 and N7 atoms comprising the bond of the covalent complex. The preference of AFB1 for specific G bases within a sequence of GC content followed that of experimental studies with the added feature that for binding to the third G base of a tetramer sequence from the 3'-end, the AFB1 displayed enhanced binding at the 3' site of the targeted guanosine. Binding of AFB1 to the second G base of a tetramer sequence from the 3'-end leads to preference for a 5' site of the targeted guanosine. Inhibition of AFB1's interaction with the targeted DNA in the presence of intercalated ethidium bromide is explained by these proposed models.     

Mapping the Binding Site of Aflatoxin B1 in DNA: Molecular Modeling of the Binding Sites for the N(7)-Guanine Adduct of Aflatoxin B1 in Different DNA Sequences

Abstract

Aflatoxin B, (AFB₁), a potent mutagen and carcinogen, forms an adduct exclusively at the N(7) position of guanine, but the structure of this adduct in double stranded DNA is not known. Molecular modeling (using the program, PSFRODO) in conjunction with molecular mechanical calculation (using the program, AMBER) are used to assess the binding modes available to this AFB₁ adduct TVvo modes appear reasonable; in one the AFB₁ moiety is intercalated between the base pair containing the adducted guanine and the adjacent base pair on the 5₁-side in reference to the adducted guanine, while in the second it is bound externally in the major groove of DNA Rotational flexibilty appears feasible in the latter providing four, potential binding sites. Molecular modeling reveals that the binding sites around the reactive guanine in different sequences are not uniformly compatible for interaction with AFB₁. As the sequence is changed, one particular external binding site would be expected to give a pattern of reactivities that is reasonably consistent with the observed sequence specificity of binding that AFB₁ shows in its reaction with DNA (Benasutti, M., Ejadi, S., Whitlow, M. D. and Loechler, E. L. (1988) Biochemistry 27, 472–481). The AFB₁ moiety is face-stacked in the major groove with its long axis approximately perpendicular to the helix axis. Favorable interactions are formed between exocyclic amino groups that project into the major groove on cytosines and adenines surrounding the reactive guanine, and oxygens in AFB₁; unfavorable interactions involve van der Waals contacts between the methyl group on thymine and the AFB₁ moiety. “Some of the sequence specificity of binding data can be rationalized more readily if it is assumed that 5′-GG-3′ sequences adopt an A-DNA structure.” Based upon molecular modeling/potential energy minimization calculation, it is difficult to predict how reactivity would change in different DNA sequences in the case of the intercalative binding mode; however, several arguments suggest that intercalation might not be favored. From these considerations a model of the structure for the transition state in reaction of AFB, with DNA is proposed involving one particular external binding site.     

Development of aflatoxin B(1)-lysine adduct monoclonal antibody for human exposure studies

Mouse monoclonal antibodies were developed against a synthetic aflatoxin B(1) (AFB)-lysine-cationized bovine serum albumin conjugate. The isotype of one of these antibodies, IIA4B3, has been classified as immunoglobulin G1(lambda). The affinity and specificity of IIA4B3 were further characterized by a competitive radioimmunoassay. The affinities of IIA4B3 for AFB and its associated adducts and metabolites are ranked as follows: AFB-lysine > 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB > AFB = 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB > aflatoxin M(1) > aflatoxin Q(1). IIA4B3 had about a 10-fold higher affinity for binding to AFB-lysine adduct than to AFB when (3)H-AFB-lysine was used as the tracer. The concentration for 50% inhibition for AFB-lysine was 0.610 pmol; that for AFB was 6.85 pmol. IIA4B3 had affinities at least sevenfold and twofold higher than those of 2B11, a previously developed antibody against parent AFB, for the major aflatoxin-DNA adducts 8,9-dihydro-8-(N(7)-guanyl)-9-hydroxy-AFB and 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl formamido)-9-hydroxy-AFB, respectively. An analytical method based on a competitive radioimmunoassay with IIA4B3 and (3)H-AFB-lysine was validated with a limit of detection of 10 fmol of AFB-lysine adduct. The method has been applied to the measurement of AFB-albumin adduct levels in human serum samples collected from the residents of areas at high risk for liver cancer.     

Accomodation of S-cis-tamoxifen-N(2)-guanine adduct within a bent and widened DNA minor groove

The non-steroidal anti-estrogen tamoxifen [TAM] has been in clinical use over the last two decades as a potent adjunct chemotherapeutic agent for treatment of breast cancer. It has also been given prophylactically to women with a strong family history of breast cancer. However, tamoxifen treatment has also been associated with increased endometrial cancer, possibly resulting from the reaction of metabolically activated tamoxifen derivatives with cellular DNA. Such DNA adducts can be mutagenic and the activities of isomeric adducts may be conformation-dependent. We therefore investigated the high resolution NMR solution conformation of one covalent adduct (cis-isomer, S-epimer of [TAM]G) formed from the reaction of tamoxifen [TAM] to N(2)-of guanine in the d(C-[TAM]G-C).d(G-C-G) sequence context at the 11-mer oligonucleotide duplex level. Our NMR results establish that the S-cis [TAM]G lesion is accomodated within a widened minor groove without disruption of the Watson-Crick [TAM]G. C and flanking Watson-Crick G.C base-pairs. The helix axis of the bound DNA oligomer is bent by about 30 degrees and is directed away from the minor groove adduct site. The presence of such a bulky [TAM]G adduct with components of the TAM residue on both the 5'- and the 3'-side of the modified base could compromise the fidelity of the minor groove polymerase scanning machinery.     

Mapping and molecular modeling of S-adenosyl-L-methionine binding sites in N-methyltransferase domains of the multifunctional polypeptide cyclosporin synthetase

We employed a highly specific photoaffinity labeling procedure, using (14)C-labeled S-adenosyl-l-methionine (AdoMet) to define the chemical structure of the AdoMet binding centers on cyclosporin synthetase (CySyn). Tryptic digestion of CySyn photolabeled with either [methyl-(14)C]AdoMet or [carboxyl-(14)C]AdoMet yielded the sequence H(2)N-Asn-Asp-Gly-Leu-Glu-Ser-Tyr-Val-Gly-Ile-Glu-Pro-Ser-Arg-COOH (residues 10644-10657), situated within the N-methyltransferase domain of module 8 of CySyn. Radiosequencing detected Glu(10654) and Pro(10655) as the major sites of derivatization. [carboxyl-(14)C]AdoMet in addition labeled Tyr(10650). Chymotryptic digestion generated the radiolabeled peptide H(2)N-Ile-Gly-Leu-Glu-Pro-Ser-Gln-Ser-Ala-Val-Gln-Phe-COOH, corresponding to amino acids 2125-2136 of the N-methyltransferase domain of module 2. The radiolabeled amino acids were identified as Glu(2128) and Pro(2129), which are equivalent in position and function to the modified residues identified with tryptic digestions in module 8. Homology modeling of the N-methyltransferase domains indicates that these regions conserve the consensus topology of the AdoMet binding fold and consensus cofactor interactions seen in structurally characterized AdoMet-dependent methyltransferases. The modified sequence regions correspond to the motif II consensus sequence element, which is involved in directly complexing the adenine and ribose components of AdoMet. We conclude that the AdoMet binding to nonribosomal peptide synthetase N-methyltransferase domains obeys the consensus cofactor interactions seen among most structurally characterized low molecular weight AdoMet-dependent methyltransferases.     

Alkylpurine-DNA-N-glycosylase excision of 7-(hydroxymethyl)-1,N6-ethenoadenine, a glycidaldehyde-derived DNA adduct

Glycidaldehyde (GDA) is a bifunctional alkylating agent that has been shown to be mutagenic in vitro and carcinogenic in rodents. However, the molecular mechanism by which it exerts these effects is not established. GDA is capable of forming exocyclic hydroxymethyl-substituted etheno adducts on base residues in vitro. One of them, 7-(hydroxymethyl)-1,N6-ethenoadenine (7-hm-epsilonA), was identified as the principal adduct in mouse skin treated with GDA or a glycidyl ether. In this work, using defined oligonucleotides containing a site-specific 7-hm-epsilonA, the human and mouse alkylpurine-DNA-N-glycosylases (APNGs), responsible for the removal of the analogous 1,N6-ethenoadenine (epsilonA) adduct, are shown to recognize and excise 7-hm-epsilonA. Such an activity can be significantly modulated by both 5' neighboring and opposite sequence contexts. The efficiency of human or mouse APNG for excision of 7-hm-epsilonA is about half that, or similar to the excision of epsilonA, respectively. When human or mouse cell-free extracts were tested, however, the extent of 7-hm-epsilonA excision is dramatically lower than that for epsilonA, suggesting that, in the crude extracts, the APNG activities toward these two adducts are differentially affected. Using cell-free extracts from APNG deficient mice, this enzyme is shown to be the primary glycosylase excising 7-hm-epsilonA. A structural approach, using molecular modeling, was employed to examine how the structure of the 7-hm-epsilonA adduct affects DNA conformation, as compared to the epsilonA adduct. These novel substrate specificities could have both biological and structural implications.     

Probing site specificity of DNA binding metallointercalators by NMR spectroscopy and molecular modeling

The molecular recognition of oligonucleotides by chiral ruthenium complexes has been probed by NMR spectroscopy using the template Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2)) (bidentate)](2+), where the bidentate ligand is one of phen (1,10-phenanthroline), dpq (dipyrido[3,2-f:2',3'-h]quinoxaline), or phi (9,10-phenanthrenequinone diimine) and picchxnMe(2)() is N,N'-dimethyl-N,N'-di(2-picolyl)-1,2-diaminocyclohexane. By varying only the bidentate ligand in a series of complexes, it was shown that the bidentate alone can alter binding modes. DNA binding studies of the Delta-cis-alpha-[Ru(RR-picchxnMe(2))(phen)](2+) complex indicate fast exchange kinetics on the chemical shift time scale and a "partial intercalation" mode of binding. This complex binds to [d(CGCGATCGCG)](2) and [d(ATATCGATAT)](2) at AT, TA, and GA sites from the minor groove, as well as to the ends of the oligonucleotide at low temperature. Studies of the Delta-cis-beta-[Ru(RR-picchxnMe(2))(phen)](2+) complex with [d(CGCGATCGCG)](2) showed that the complex binds only weakly to the ends of the oligonucleotide. The interaction of Delta-cis-alpha-[Ru(RR-picchxnMe(2))(dpq)](2+) with [d(CGCGATCGCG)](2) showed intermediate exchange kinetics and evidence of minor groove intercalation at the GA base step. In contrast to the phen and dpq complexes, Delta-cis-alpha- and Delta-cis-beta-[Ru(RR-picchxnMe(2))(phi)](2+) showed evidence of major groove binding independent of the metal ion configuration. DNA stabilization induced by complex binding to [d(CGCGATCGCG)](2) (measured as DeltaT(m)) increases in the order phen < dpq and DNA affinity in the order phen < dpq < phi. The groove binding preferences exhibited by the different bidentate ligands is explained with the aid of molecular modeling experiments.     

The N2-guanine adduct but not the C8-guanine or N6-adenine adducts formed by 4-nitroquinoline 1-oxide blocks the 3'-5' exonuclease action of T4 DNA polymerase

When O-acetyl-4-(hydroxyamino)quinoline 1-oxide (Ac-4HAQO) reacts with double-stranded DNA at 37 degrees C the major products, N2-guanine, C8-guanine, and N6-adenine adducts, are formed in the proportions of 5:3:2, respectively. When the reaction is carried out with single-stranded DNA at 0 degree C, the products are found in the ratio 1:7:2. Unique 174-bp DNA fragments were modified in these ways and used as substrates for the 3'-5' exonuclease activity of T4 DNA polymerase. The results obtained showed that the exonuclease is blocked by the N2-guanine adduct but not the other two adducts. Interpretation of the cleavage patterns suggested that the enzyme stopped 2 nucleotides before the N2-guanine adduct. The N2-guanine adduct lies in the minor groove of the DNA double helix, while the other two adducts are found in the major groove. Apparently, only the former hinders progression of the enzyme.     

Mapping the binding site on small ankyrin 1 for obscurin

Small ankyrin 1 (sAnk1), an integral protein of the sarcoplasmic reticulum encoded by the ANK1 gene, binds with nanomolar affinity to the C terminus of obscurin, a giant protein surrounding the contractile apparatus in striated muscle. We used site-directed mutagenesis to characterize the binding site on sAnk1, specifically addressing the role of two putative amphipathic, positively charged helices. We measured binding qualitatively by blot overlay assays and quantitatively by surface plasmon resonance and showed that both positively charged sequences are required for activity. We showed further that substitution of a lysine or arginine with an alanine or glutamate located at the same position along either of the two putative helices has similar inhibitory or stimulatory effects on binding and that the effects of a particular mutation depended on the position of the mutated amino acid in each helix. We modeled the structure of the binding region of sAnk1 by homology with ankyrin repeats of human Notch1, which have a similar pattern of charged and hydrophobic residues. Our modeling suggested that each of the two positively charged sequences forms pairs of amphipathic, anti-parallel alpha-helices flanked by beta-hairpin-like turns. Most of the residues in homologous positions along each helical unit have similar, though not identical, orientations. CD spectroscopy confirmed the alpha-helical content of sAnk1, approximately 33%, predicted by the model. Thus, structural and mutational studies of the binding region on sAnk1 for obscurin suggest that it consists of two ankyrin repeats with very similar structures.     

Depurination from DNA of 7-methylguanine, 7-(2-aminoethyl)-guanine and ring-opened 7-methylguanines

DNA was reacted with dimethyl sulphate and ethyleneimine to afford respective 7-methylguanine and 7-(2-aminoethyl)guanine derivatives. The substituted DNA was boiled in 0.1 M NaCl containing 10 mM phosphate buffer (pH 7.0), and the release of 7-alkylguanines, guanine and adenine was followed. The half-lives of depurination were 1.5 and 4.1 min for 7-(2-aminoethyl)guanine and 7-methylguanine, respectively. 7-Methylguanine was released some 60 times faster than guanine and adenine. When 7-methylguanine-containing DNA was treated in alkali to cause imidazole ring-opening, two products were liberated by boiling the DNA solution. These products were released with apparent half-lives of 69 and 34 min. These ring-opened products isomerized to each other completely within 1 h at 37°C. The isomers had an identical ultraviolet spectrum and they displayed a pK_a of 9.8. When silylated and analysed in gas chromatography-mass spectroscopy the two isomers had an identical molecular weight and fragmentation pattern, consistent with a structural assignment as N⁵-methyl-N⁵-formyl-2,5,6-triamino-4-oxopyrimidine. Only one of the isomers appeared to be present on DNA; the isomerization took place when the ring-opened product was released into solution.     

Efficient and error-free replication past a minor-groove N2-guanine adduct by the sequential action of yeast Rev1 and DNA polymerase zeta

Rev1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase zeta (Pol zeta). Rev1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While Rev1 plays an indispensable structural role in Pol zeta-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, Rev1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N(2)-adducted guanines in DNA. To test this idea, we examined whether Rev1 could incorporate a C opposite the gamma-hydroxy-1,N(2)-propano-2'deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N(2) of guanine. Acrolein, an alpha,beta-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that Rev1 efficiently incorporates a C opposite this adduct from which Pol zeta subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the Rev1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N(2)-guanine DNA minor-groove adducts that form in DNA.     

Wobble dC.dA pairing 5' to the cationic guanine N7 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct: implications for nontargeted AFB1 mutagenesis

The structure of 5'-d(ACATC(AFB)GATCT)-3'.5'-d(AGATCAATGT)-3', containing the C(5).A(16) mismatch at the base pair 5' to the modified (AFB)G(6), was determined by NMR. The characteristic 5'-intercalation of the AFB(1) moiety was maintained. The mismatched C(5).A(16) pair existed in the wobble conformation, with the C(5) imino nitrogen hydrogen bonded to the A(16) exocyclic amino group. The wobble pair existed as a mixture of protonated and nonprotonated species. The pK(a) for protonation at the A(16) imino nitrogen was similar to that of the C(5).A(16) wobble pair in the corresponding duplex not adducted with AFB(1). Overall, the presence of AFB(1) did not interfere with wobble pair formation at the mismatched site. Molecular dynamics calculations restrained by distances derived from NOE data and torsion angles derived from (1)H (3)J couplings were carried out for both the protonated and nonprotonated wobble pairs at C(5).A(16). Both sets of calculations predicted the A(16) amino group was within 3 A of the C(5) imino nitrogen. The calculations suggested that protonation of the C(5).A(16) wobble pair should shift C(5) toward the major groove and shift A(16) toward the minor groove. The NMR data showed evidence for the presence of a minor conformation characterized by unusual NOEs between T(4) and (AFB)G(6). T(4) is two nucleotides in the 5'-direction from the modified base. These NOEs suggested that in the minor conformation nucleotide T(4) was in closer proximity to (AFB)G(6) than would be expected for duplex DNA. Modeling studies examined the possibility that T(4) transiently paired with the mismatched A(16), allowing it to come within NOE distance of (AFB)G(6). This model structure was consistent with the unusual NOEs associated with the minor conformation. The structural studies are discussed in relationship to nontargeted C --> T transitions observed 5' to the modified (AFB)G in site-specific mutagenesis experiments [Bailey, E. A., Iyer, R. S., Stone, M. P., Harris, T. M., and Essigmann, J. M. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1535-1539].     

Effects of benzo[a]pyrene adduct stereochemistry on downstream DNA replication in vitro: evidence for different adduct conformations within the active site of DNA polymerase I (Klenow fragment)

The presence of bulky adducts in DNA is known to interfere with DNA replication not only at the site of the lesion but also at positions up to 5 nucleotides past the adduct location. Kinetic studies of primer extension by exonuclease-deficient E. coli DNA polymerase I (Klenow fragment) (KF) when (+)-trans- or (+)-cis-B[a]P-N(2)-dG adducts were positioned in the double-stranded region of the primer-templates showed that both stereoisomers significantly block downstream replication. However the (+)-cis adduct, which causes a stronger inhibition of the nucleotides insertion across from and immediately past the lesion, affected the downstream replication to a much smaller extent than did the (+)-trans adduct, especially when the B[a]P-modified dG was properly paired with a dC. The effects of mismatches across from the adduct and the sequence context surrounding the adduct were also dependent on the stereochemistry of the B[a]P adduct. Thus, the identity of the nucleotide across from the adduct that provided the best downstream replication was different for the (+)-cis and (+)-trans adducts, a factor that might differentially contribute to the mutagenic bypass of these lesions. These findings provide strong direct evidence that the conformations of the (+)-cis and (+)-trans adducts within the active site of KF are significantly different and probably differentially affect the interactions of the polymerase with the minor groove, thereby leading to different replication trends. The stereochemistry of the adduct was also found to differentially affect the sequence-mediated primer-template misalignments, resulting in different consequences during the bypass of the lesion.     

Mapping the UDP-glucuronic acid binding site in UDP-glucuronosyltransferase-1A10 by homology-based modeling: confirmation with biochemical evidence

The UDP-glucuronosyltransferase (UGT) isozyme system is critical for protecting the body against endogenous and exogenous chemicals by linking glucuronic acid donated by UDP-glucuronic acid to a lipophilic acceptor substrate. UGTs convert metabolites, dietary constituents, and environmental toxicants to highly excretable glucuronides. Because of difficulties associated with purifying endoplasmic reticulum-bound UGTs for structural studies, we carried out homology-based computer modeling to aid analysis. The search found structural homology in Escherichia coli UDP-galactose 4-epimerase. Consistent with predicted similarities involving the common UDP moiety in substrate/inhibitor, UDP-glucose and UDP-hexanol amine caused competitive inhibition by Lineweaver-Burk plots. Among predicted binding sites N292, K314, K315, and K404 in UGT1A10, two informative sets of mutants K314R/Q/A/E/G and K404R/E had null activities or 2.7-fold higher/50% less activity, respectively. Scatchard analysis of binding data of the affinity ligand, 5-azidouridine-[beta- (32)P]diphosphoglucuronic acid, to purified UGT1A10-His or UGT1A7-His revealed high- and low-affinity binding sites. 2-Nitro-5-thiocyanobenzoic acid-digested UGT1A10-His bound with the radiolabeled affinity ligand revealed an 11.3 and 14.3 kDa peptide associated with K314 and K404, respectively, in a discontinuous SDS-PAGE system. Similar treatment of 1A10His-K314A bound with the ligand lacked both peptides; 1A10-HisK404R- and 1A10-HisK404E showed 1.3-fold greater and 50% less label in the 14.3 kDa peptide, respectively, compared to 1A10-His without affecting the 11.3 kDa peptide. Scatchard analysis of binding data of the affinity ligand to 1A10His-K404R and -K404E showed a 6-fold reduction and a large increase in K d, respectively. Our results indicate that K314 and K404 are required UDP-glcA binding sites in 1A10, that K404 controls activity and high-affinity sites, and that K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321, equivalent to K314, in UGT2B15 and 2B17 and I321 in the inactive UGT8, which suggests UGT2B15 and 2B17 contain suboptimal activity. Hence our data strongly support UDP-glcA binding to K314 and K404 in UGT1A10.     

Palladium(II) binding to N(7) of acyclovir: DNA interaction and herpes simplex virus (HSV-1) inhibitory activity

Cytotoxicity and herpes simplex virus (HSV-1) inhibitory activity of acyclovir (ACV), 9-[(2-hydroxyethoxy)methyl]guanine, and the palladium(II) coordination complex cis-[PdCl₂(H₂O)(N7-ACV)] · ACV · xH₂O have been tested in African green monkey kidney (Vero line) epithelial cell cultures. The N(7) position of ACV represents the preferred binding site to afford a pseudo-chelate N7/O6 Pd(II) complex involving H-bonds with the cis H₂O molecule. The Pd(II)-ACV complex has been structurally characterized by FTIR and ¹H NMR spectroscopy techniques, chemical composition was measured by elemental analysis, and the thermoanalytical study was performed by TG/DTA. The recognition of secondary ACV molecules by the Pd(II) derivative promotes cooperatively potent HSV-1 inhibitory activity which, in turn, strongly depends on concentration conditions. At the optimal concentration of 10 μM, this complex exhibits antiviral efficiency in vitro, approximately hundred-fold (ca. 1.87 log₁₀) more effective in herpes-infected cells when compared with that of the parent ACV molecules. The molecular-level observation of noticeable modifications caused by the complex on the morphology of the plasmid pBR322 DNA was monitored by AFM, whose mutual interaction evolves to eventually afford DNA condensates upon increasing the period of incubation.     

Characterization of the aflatoxin B1-binding site of rat albumin

A fluorescence-enhancement method was used to investigate the non-covalent interaction between aflatoxin B1 and rat albumin. Solvent-induced shifts in the emission spectrum of aflatoxin B1 provided evidence that the aflatoxin B1-binding site of rat albumin is a highly nonpolar environment. A dissociation constant of 20 microM was determined at 20 degrees C. The possibility that aflatoxin B1 binds one of the three major drug sites of albumin was investigated by ligand-displacement experiments. Mechanisms whereby marker ligands displace aflatoxin B1 were further investigated by comparing the experimental binding parameters with those derived theoretically, assuming competitive binding. The results indicate that: aflatoxin B1 and phenylbutazone compete for a common high-affinity site on rat albumin; high-affinity binding of aflatoxin B1 and site-II marker ligands takes place independently; aflatoxin B1 does not compete with either cholate or warfarin for the same high-affinity site, but the simultaneous binding of warfarin or cholate negatively modulates the binding of aflatoxin B1 to albumin. Fluorescence energy-transfer studies show that the lone tryptophan residue, Trp-214, is not associated with the aflatoxin B1-binding site.     

Abasic sites in duplex DNA: molecular modeling of sequence-dependent effects on conformation

Molecular modeling calculations using JUnction Minimization of Nucleic Acids (JUMNA) have been used to study sequence effects on the conformation of abasic sites within duplex DNA. We have considered lesions leading to all possible unpaired bases (X), adenine, guanine, cytosine, or thymine contained within two distinct sequence contexts, CXC and GXG. Calculations were carried out on DNA 11-mers using extensive conformational search techniques to locate the most stable abasic conformations and using Poisson-Boltzmann corrected electrostatics to account for solvation effects. The results, which are in very good agreement with available experimental data, point to strong sequence effects on both the position of the unpaired base (intra or extrahelical) and on the overall curvature induced by the abasic lesion. For CXC, unpaired purines are found to lie within the helix, while unpaired pyrimidines are either extrahelical or in equilibrium between the intra and extrahelical forms. For GXG, all unpaired bases lead to intrahelical forms, but with marked, sequence-dependent differences in induced curvature.