Cross-linking of the fingers subdomain of human immunodeficiency virus type 1 reverse transcriptase to template-primer.

Cross-linking experiments were performed with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Two approaches were used--photoaffinity cross-linking and disulfide chemical cross-linking (using an oligonucleotide that contained an N(2)-modified dG with a reactive thiol group). In the former case, cross-linking can occur to any nucleotide in either DNA strand, and in the latter case, a specific cross-link is produced between the template and the enzyme. Neither the introduction of the unique cysteine residues into the fingers nor the modification of these residues with photocross-linking reagents caused a significant decrease in the enzymatic activities of RT. We were able to use this model system to investigate interactions between specific points on the fingers domain of RT and double-stranded DNA (dsDNA). Photoaffinity cross-linking of the template to the modified RTs with Cys residues in positions 65, 67, 70, and 74 of the fingers domain of the p66 subunit was relatively efficient. Azide-modified Cys residues produced 10 to 25% cross-linking, whereas diazirine modified residues produced 5 to 8% cross-linking. Disulfide cross-linking yields were up to 90%. All of the modified RTs preferentially photocross-linked to the 5' extended template strand of the dsDNA template-primer substrate. The preferred sites of interactions were on the extended template, 5 to 7 bases beyond the polymerase active site. HIV-1 RT is quite flexible. There are conformational changes associated with substrate binding. Cross-linking was used to detect intramolecular movements associated with binding of the incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreases the efficiency of cross-linking, but causes only modest changes in the preferred positions of cross-linking. This suggests that the interactions between the fingers of p66 and the extended template involve the "open" configuration of the enzyme with the fingers away from the active site rather than the closed configuration with the fingers in direct contact with the incoming dNTP. This experimental approach can be used to measure distances between any site on the surface of the protein and an interacting molecule.

Patterns of resistance to exonuclease digestion of oligonucleotides containing polycyclic aromatic hydrocarbon diol epoxide adducts at N6 of deoxyadenosine.

The effect of adduct stereochemistry on the susceptibility to hydrolysis by snake venom (VPD) and bovine spleen (SPD) phosphodiesterases was investigated with short deoxyoligonucleotides containing defined adducts derived from alkylation of the exocyclic 6-amino group of dA by polycyclic aromatic hydrocarbon diol epoxides (DEs). In accordance with several earlier reports, we have found that adducts with R configuration at the site of attachment of dA to the DE moiety derived from either benzo[a]pyrene (BaP) or benzo[c]phenanthrene (BcPh) are generally more resistant to hydrolysis by VPD than are their (S)-diastereomers. The reaction with VPD initially yields a fragment containing the adducted dA residue at its 3'-end, which slowly hydrolyzes to a dimer (pXpA*) with an intact 5'-phosphodiester bond to the adducted dA. With several of the adducts studied, this dimer underwent cleavage to release eventually the monomeric adduct p(dA*). Adducts derived from cis opening of the epoxide ring of both BaP and BcPh DEs were considerably more resistant to VPD than the corresponding trans-opened adducts. Although several previous investigations had suggested that oligonucleotides containing adducts which have S configuration at the site of attachment of the hydrocarbon to adenine are more resistant to cleavage by SPD than are their (R)-diastereomers, the present results with a more extensive set of oligonucleotides indicate that SPD, in contrast to VPD, exhibits little discrimination between adducts with R and S configuration at the site of attachment to the base. Notably, for both enzymes, the most resistant internucleotide linkage (the bond 3'-sugar to phosphate for VPD and 5'-sugar to phosphate for SPD) is between the modified base and the base immediately 5' to it, regardless of the configuration of the adduct.

Effect of benzo-ring hydroxyl groups on site-specific mutagenesis by tetrahydrobenzo[a]pyrene adducts at N(6) of deoxyadenosine.

We have previously investigated the mutations induced on replication in Escherichia coli of the M13mp7L2 genome containing each of the eight possible adducts derived from the four optically active 7,8-diol 9,10-epoxide metabolites of benzo[a]pyrene (B[a]P) by alkylation of a specific deoxyadenosine (dAdo) residue at N(6). Observed mutational frequencies depended in part on the relative spatial orientations of the three hydroxyl groups in these adducts. To determine how the presence or absence of these hydroxyl groups affects mutational response, we have synthesized 16-mer oligonucleotides with the same sequence as one of those previously studied with the diol epoxide adducts, but containing B[a]P-dAdo adducts in which two or all three of the adduct hydroxyl groups were replaced by hydrogen. Transfection of the adducted M13 constructs into SOS-induced Escherichia coli consistently gave fewer infective centers than the control construct, with viabilities ranging from 8.4 to 44.9% relative to control. In general, decreasing the number of adduct hydroxyls decreased the total frequency of substitution mutations induced. For all but one of the present adducts, the total mutational frequency was lower than that for any of the previously reported diol epoxide adducts in the same sequence. Remarkably, this (9S,10R)-adduct with cis orientation of the dAdo residue and the 9-OH group gave the highest mutational frequency of all the B[a]P adducts studied in this sequence, including the diol epoxide adducts. With the present adducts, A --> T transversions predominated, with smaller numbers of A --> G transitions and even fewer A --> C transversions.

New insights on the mechanisms of the pH-independent reactions of benzo[a]pyrenes 7,8-diol 9,10-epoxides.

The rates and products of the reactions of (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (1) and (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (2) in water and dioxane-water mixtures have been determined over a pH range wider than that of earlier studies. This study provides additional insight on the mechanisms of the pH-independent reactions of 1 and 2. The rate profile for reaction of 1 shows acid-catalyzed hydrolysis at pH <5, a rate plateau at pH 5-9.5, a negative inflection at pH 10-11.5, and a rate increase at pH >11.5. The rate decrease between pH 10 and pH 11.5 is accompanied by a decrease in the yield of tetrols from 60% (pH 8) to 29% (pH 11.2) and is interpreted to be the result of a partial change in mechanism brought about by attack of hydroxide ion acting as a base to deprotonate a carbocation intermediate and regenerate 1 at pH >10, thus reducing the contribution of the pathway for tetrol formation in which water attacks the carbocation. The rate profile for the reaction of 2 exhibits only a single rate plateau at intermediate pH, along with increases in rate at low and high pH because of second-order reactions of 2 with H+ and HO-, respectively. The lack of a rate depression at pH >10 and the product studies for the reaction of 2 in dilute sodium azide solutions suggest that the tetrol-forming reactions of the pH-independent reaction of 2 are concerted or near-concerted.

O(6)-allyl protected deoxyguanosine adducts of polycyclic aromatic hydrocarbons as building blocks for the synthesis of oligonucleotides.

We describe a synthetic strategy for the preparation of oligonucleotides using N(2)-alkylated and O(6)-allyl protected deoxyguanosine phosphoramidite building blocks derived from cis- and trans-opened (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and from trans-opened (+/-)-3alpha,4beta-dihydroxy-1alpha,2alpha-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene. The appropriately blocked phosphoramidite building blocks were obtained as mixtures of the cis- and trans-opened diol epoxide adducts upon initial reaction of the diol epoxides with O(6)-allyl-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine. Key to the present approach is the removal of the O(6)-allyl protecting group utilizing a palladium catalyst prior to release of the constructed oligonucleotide with ammonia from the solid support. The methodology described enables a very convenient access to oligonucleotides containing cis- and trans-N(2)-deoxyguanosine adducts of polycyclic aromatic hydrocarbons in different sequence contexts.

Differences between the mutational consequences of replication of cis- and trans-opened benzo[a]pyrene 7,8-diol 9,10-epoxide-deoxyguanosine adducts in M13mp7L2 constructs.

The four adducts at N(2) of deoxyguanosine derived from cis-opening at C-10 of four optically active isomers of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene were incorporated into 5'-TTCGAATCCTTCCCCC [context III(G)] and 5'-GGGGTTCCCGAGCGGC [context IV(G)] at the underlined site. The mutagenic consequences of these lesions in each of the two sequence contexts were examined after ligation of the modified oligonucleotides into single-stranded M13mp7L2 and replication of the vector in SOS-induced Escherichia coli. Total frequencies of base substitution mutations ranged between 14 and 48%. The mutation frequencies were generally higher in context IV(G) than in context III(G), and consisted mainly of G-->T followed by G-->C base substitutions. A substantial number of deletions or insertions of one guanine was also found for all adducts in context IV(G), where the adduct is located at the 3'-end of a run of five guanines. The overall frequencies of base substitution mutations induced by cis-opened adducts were substantially higher than those observed with the trans-opened dGuo adducts in the same sequences [Page et al. (1998) Biochemistry 37, 9127-9137]. Although G-->T base substitutions predominated for both the cis- and trans-opened adducts, the cis-opened dGuo adducts generally resulted in a higher proportion of G-->C [particularly in context III(G)] relative to G-->A, whereas the opposite was true for the trans-opened dGuo adducts. The present results along with previous data indicate that mutagenicity is highly dependent on a combination of sequence context and adduct stereochemistry.

Solution structure of a trans-opened (10S)-dA adduct of (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a fully complementary DNA duplex: evidence for a major syn conformation.

Two-dimensional NMR was used to determine the solution structure of an undecanucleotide duplex, d(CGGTCACGAGG).d(CCTCGTGACCG), in which (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is covalently bonded to the exocyclic N(6)() amino group of the central deoxyadenosine, dA(6), through trans addition at C10 of the epoxide (to give a 10S adduct). The present study represents the first NMR structure of a benzo[a]pyrene (10S)-dA adduct in DNA with a complementary T opposite the modified dA. Exchangeable and nonexchangeable protons of the modified duplex were assigned by the use of TOCSY (in D(2)O) and NOESY spectra (in H(2)O and D(2)O). Sequential NOEs expected for a B-type DNA conformation with typical Watson-Crick base pairing are observed along the duplex, except at the lesion site. We observed a strong intraresidue NOE cross-peak between H1' and H8 of the modified dA(6). The sugar H2' and H2' ' of dC(5) lacked NOE cross-peaks with H8 of dA(6) but showed weak interactions with H2 of dA(6) instead. In addition, the chemical shift of the H8 proton (7.51 ppm) of dA(6) appears at a higher field than that of H2 (8.48 ppm). These NOE and chemical shift data for the dA(6) base protons are typical of a syn glycosidic bond at the modified base. Restrained molecular dynamics/energy minimization calculations show that the hydrocarbon is intercalated from the major groove on the 3'-side of the modified base between base pairs A(6)-T(17) and C(7)-G(16) and confirm the syn glycosidic angle (58 degrees ) of the modified dA(6). In the syn structure, a weak A-T hydrogen bond is possible between the N3-H proton of T(17) and N7 of dA(6) (at a distance of 3.11 A), whereas N1, the usual hydrogen bonding partner for N3-H of T when dA is in the anti conformation, is 6.31 A away from this proton. The 10(S)-dA modified DNA duplex remains in a right-handed helix, which bends in the direction of the aliphatic ring of BaP at about 42 degrees from the helical axis. ROESY experiments provided evidence for interconversion between the major, syn conformer and a minor, possibly anti, conformer.

Tumorigenicity of racemic and optically pure bay region diol epoxides and other derivatives of the nitrogen heterocycle dibenz[a,h]acridine on mouse skin.

CD-1 female mice were initiated with a single topical application of 500 nmol dibenz[a,h]acridine (DB[a,h]Acr), its racemic trans-1,2-, 3,4-, 8,9- and 10,11-dihydrodiols, racemic DB[a,h]Acr 3,4-diol 1,2-epoxide-1 and -2 or racemic DB[a,h]Acr 10,11-diol 8,9-epoxide-1 and -2, where the benzylic hydroxyl group is either cis (isomer 1) or trans (isomer 2) to the epoxide oxygen. The mice were subsequently treated twice weekly with 12-O-tetradecanoylphorbol 13-acetate for 25 weeks. High tumorigenicity was observed only for DB[a,h]Acr, its 10,11-dihydrodiol and DB[a,h]Acr 10,11-diol 8,9-epoxide-2 (3.3, 1.2 and 1.6 tumors/mouse, respectively). The tumor-initiating activity of a 50 nmol dose of DB[a,h]Acr and the optically active (+)- and (-)-enantiomers of DB[a,h]Acr 10,11-dihydrodiol and of the optically active DB[a,h]Acr 10,11-diol 8,9-epoxide-1 and -2 were also studied. Only DB[a,h]Acr, (-)-DB[a,h]Acr (10R,11R)-dihydrodiol and the bay region (+)-(8R,9S,10S,11R)-diol epoxide-2 were highly active (1.6, 1.7 and 2.4 tumors/mouse, respectively). These results are consistent with previous studies which showed that the corresponding bay region RSSR diol epoxides of benzo[a]pyrene, benz[a]anthracene, chrysene and benzo[c]phenanthrene as well as the aza-polycyclic dibenz[c,h]acridine are the most tumorigenic isomers.

Solvent-free synthesis of benzo[a]pyrene 7,8-diol 9,10-epoxide adducts at the N(2)-position of deoxyguanosine.

[reaction: see text] The first solid-state (or solvent-free) synthesis of protected deoxyguanosine (dG) adducts of benzo[a]pyrene diol epoxides at room temperature is reported. Whereas dG adducts derived from cis- and trans-opening of (+/-)-7beta,8alpha-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (DE-1 1) are formed as a 1:1 mixture, the direct opening of the diastereomeric (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (DE-2, 2) produced a 15:85 ratio favoring the trans-opened dG adduct 7.

Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2.

The adduct that would arise from cis opening of (+)-(1S,2R,3R, 4S)-3,4-dihydroxy-1,2-epoxy-benzo[c]phenan-threne (benzo[c]phenanthrene diol epoxide-2, where the benzylic hydroxyl group and the epoxide oxygen are trans) by the exocyclic N6-amino group of deoxyadenosine was incorporated at the marked site into four oligonucleotides, 5'-CAGA*TTTAGAGTCTGC-3', 5'-CAGTGCAGA*TTTAGAG-3', 5'-GTGCAGA*TTTAGA-3' and 5'-TGCAGA*TTTA-3'. The oligonucleotides were inserted into M13mp7L2 and the vector transfected into SOS-induced Escherichia coli SMH77 which were then plated on agar plates. The experiments reported here were designed to test the effect of the lesion position (the marked A in the sequences above) on the ligation efficiency of the insert and the frequency of failed constructs, as well as any possible effects on the mutagenic consequences of the lesion. The construct survival was estimated from the number of plaques formed following transformation, and mutation frequencies were estimated from sequencing of randomly picked plaques. Moving the adduct site to the middle of the sequence increased considerably the ligation efficiency regardless of the length of the inserted oligonucleotide, and changing the insert length or the adduct location did not markedly affect the frequency (40-58.6%) or distribution of mutations observed. Thus, so long as the local sequence (five or six bases surrounding the adduct) remains constant, the size of the oligonucleotide insert and the position of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.

Dose-dependent differences in the profile of mutations induced by carcinogenic (R,S,S,R) bay- and fjord-region diol epoxides of polycyclic aromatic hydrocarbons.

Chinese hamster V79 cells were exposed to a high or low concentration of the highly carcinogenic (R,S,S,R) or the less active (S,R,R,S) bay- or fjord-region diol epoxides of benzo[a]pyrene, benzo[c]phenanthrene or dibenz[c,h]acridine. Independent 8-azaguanine-resistant clones were isolated, and base substitutions at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus were determined. For the three (R,S,S,R) diol epoxides studied, the proportion of mutations at AT base pairs increased as the concentration of diol epoxide decreased. Concentration-dependent differences in the mutational profile were not observed, however, for the three (S,R,R,S) diol epoxides. In studies, with V-H1 cells (a DNA repair deficient variant of V79 cells), a concentration-dependent difference in the profile of mutations for the (R,S,S,R) diol epoxide of benzo[a]pyrene was not observed. These results suggest that concentration-dependent differences in the mutational profile are dependent on an intact DNA repair system. In additional studies, we initiated mouse skin with a high or low dose of benzo[a]pyrene and promoted the mice for 26 weeks with 12-O-tetradecanoylphorbol-13-acetate. Papillomas were examined for mutations in the c-Ha-ras proto-oncogene. Dose-dependent differences in the profile of c-Ha-ras mutations in the tumors were observed. In summary, (i) dose-dependent differences in mutational profiles at the hprt locus were observed in Chinese hamster V79 cells treated with several highly mutagenic and carcinogenic (R,S,S,R) bay- or fjord-region diol epoxides but not with their less active (S,R,R,S) diol epoxide enantiomers, (ii) a dose-dependent difference in the mutational profile was not observed for the (R,S,S,R) diol epoxide of benzo[a]pyrene in a DNA-repair defective V79 cell line, and (iii) a dose-dependent difference in the mutational profile in the c-Ha-ras proto-oncogene was observed in tumors from mice treated with a high or low dose of benzo[a]pyrene.

HPLC-MS/MS identification of positionally isomeric benzo[c]phenanthrene diol epoxide adducts in duplex DNA.

LC-MS and LC-MS/MS analyses were used to investigate the chemoselectivity of the carcinogenic diol epoxide metabolite, (-)-(1R,2S,3S,4R)-1,2-epoxy-3,4-dihydroxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene [(-)-(R,S,S,R)-BcPh DE-2], on reaction in vitro with an oligonucleotide dodecamer derived from the HPRT gene. The sequence of this dodecamer, 5'-T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12)-3', contains a base (corresponding to A(7)) which is a hot spot for mutagenesis in the hprt gene induced by the carcinogenic (R,S,S,R)-enantiomer of benzo[a]pyrene 7,8-diol 9,10-epoxide, and an adjacent base (corresponding to A(6)) which gave no mutations with this diol epoxide. Modified oligonucleotides were generated by reaction of (-)-BcPh DE-2 with both the single-stranded and duplex forms of the dodecamer. Multiple purine targets in both strands led to the formation of complex reaction mixtures of regioisomeric BcPh DE-modified oligonucleotides, which were partially separated by reverse phase HPLC on a polystyrene-divinylbenzene column. On-line LC-MS data allowed facile distinction between adducts on the two strands of the duplex, and MS/MS analysis permitted unambiguous assignment of the major sites of modification in the regioisomeric, adducted strands. In the duplex, these sites were at A(6), A(7), and G(8). Interestingly, the "hot spot" A(7)w as about 3 times more reactive with the BcPh DE than the "cold spot" A(6). Adduct formation from the single-stranded dodecamer was less selective, and resulted in more extensive alkylation of G residues.

Tumorigenicity of four optically active bay-region 3,4-diol 1, 2-epoxides and other derivatives of the nitrogen heterocycle dibenz[c,h]acridine on mouse skin and in newborn mice.

The nitrogen heterocycle dibenz[c,h]acridine (DB[c,h]ACR) and the enantiomers of the diastereomeric pair of bay-region 3,4-diol 1, 2-epoxides as well as other bay-region epoxides and dihydrodiol derivatives of this hydrocarbon have been evaluated for tumorigenicity on mouse skin and in the newborn mouse. On mouse skin, a single topical application of 50 or 200 nmol of compound was followed 10 days later by twice-weekly applications of the tumor promoter 12-O:-tetradecanoylphorbol-13-acetate for 20 weeks. DB[c, h]ACR and the four optically pure, bay-region 3,4-diol-1,2-epoxide isomers all had significant tumor- initiating activity. The isomer with (1R,2S,3S,4R) absolute configuration [(+)-DE-2] was the most active diol epoxide isomer. The (-)-(3R,4R)-dihydrodiol of DB[c, h]ACR, the expected metabolic precursor of the bay-region (+)-DE-2, was 4- to 6-fold more tumorigenic than its corresponding (+)-enantiomer. In tumorigenicity studies in newborn mice, a total dose of 70-175 nmol of DB[c,h]ACR or one of its derivatives was injected i.p. on days 1, 8 and 15 of life, and tumorigenic activity was determined when the mice were 36-39 weeks old. DB[c,h]ACR produced a significant number of pulmonary tumors and also produced hepatic tumors in male mice. Of the four optically active bay-region diol epoxides, only (+)-DE-2 and (+)-DE-1 with (1R,2S,3S,4R) and (1S, 2R,3S,4R) absolute configuration, respectively, produced a significant tumor incidence. At an equivalent dose, the (+)-DE-2 isomer produced several-fold more pulmonary tumors and hepatic tumors than the (+)-DE-1 isomer. The (-)-(3R,4R)-dihydrodiol, metabolic precursor of the bay-region (+)-DE-2, was strongly active and induced an equal number of pulmonary and hepatic tumors as did DB[c,h]ACR. The (+)-(3S,4S) dihydrodiol was less active. The bay-region (+)-(1R,2S)-epoxide of 1,2,3,4-tetrahydro DB[c,h]ACR was strongly tumorigenic in newborn mice whereas its (-)-(1S, 2R)-enantiomer was inactive. This contrasts with the data on mouse skin where both enantiomers had substantial tumorigenic activity. In summary, the bay-region (+)-(1R,2S,3S,4R)-3,4-diol 1,2-epoxide of DB[c,h]ACR was the most tumorigenic of the four optically active bay-region diol epoxides of DB[c,h]ACR on mouse skin and in the newborn mouse. These results with a nitrogen heterocycle are similar to earlier data indicating high tumorigenic activity for the R,S,S,R bay-region diol epoxides of several carbocyclic polycyclic aromatic hydrocarbons.

NMR evidence for syn-anti interconversion of a trans opened (10R)-dA adduct of benzo[a]pyrene (7S,8R)-diol (9R,10S)-epoxide in a DNA duplex.

2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12))-d(T(13)G(14)C( 15)C(16)C(17)T(18)T(19)G(20)A(21)C(22)T(23)A(24)), containing a 10R adduct at dA(7) that corresponds to trans addition of the N(6)-amino group of dA(7) to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5'-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S, R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.

Marked differences in base selectivity between DNA and the free nucleotides upon adduct formation from Bay- and Fjord-region diol epoxides.

Distributions of adducts formed from each of the four optically active isomers of 3,4-dihydroxy-1,2-epoxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene and of 7,8-dihydroxy-9,10-epoxy-7,8, 9,10- tetrahydrobenzo[a]pyrene (BcPh and BaP diol epoxides) on reaction with an equimolar mixture of deoxyadenosine and deoxyguanosine 5'-monophosphates were compared with the known adduct distributions from these diol epoxides (DEs) upon reaction with calf thymus DNA in vitro. In the presence of an equimolar (100 mM total) mixture of dAMP and dGMP, the efficiency of formation of all types of adducts relative to tetraols is comparable for both the BaP ( approximately 40-60%) and BcPh ( approximately 30-40%) diol epoxides. This is in contrast to the partitioning between tetraols and adducts observed with DNA, where the BcPh DEs form adducts much more efficiently than the BaP DEs. Preference for trans versus cis ring opening by the exocyclic amino groups of the free nucleotides in the dAMP/dGMP mixture is greater for the DE diastereomer in which the benzylic hydroxyl group and the epoxide oxygen are trans (DE-2). This is qualitatively similar to the preferences for trans versus cis adduct formation on reaction of these isomers with DNA, as well as trans versus cis tetraol formation on their acid hydrolysis. For the BcPh DE isomers, competitive reaction between dGMP and dAMP gives 40-62% of the total exocyclic amino group adducts as dA adducts. A similar distribution of dG versus dA adducts had previously been observed on reaction of the BcPh DEs with DNA, except in the case of (+)-3(R),4(S)-dihydroxy-1(R),2(S)-epoxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene, which gives approximately 85% dA adducts on reaction with DNA. With the BaP DEs, 60-77% of the exocyclic amino group adducts formed upon competitive reaction with the free nucleotides are derived from dGMP. The observed dG selectivity of these BaP DEs is much smaller with the nucleotide mixture than it is with DNA, leading to the conclusion that DNA structure has a much larger modifying effect on the base selectivity of the BaP relative to the BcPh DEs.

Position-specific trapping of topoisomerase I-DNA cleavage complexes by intercalated benzo[a]- pyrene diol epoxide adducts at the 6-amino group of adenine.

DNA topoisomerase I (top1) is the target of potent anticancer agents, including camptothecins and DNA intercalators, which reversibly stabilize (trap) top1 catalytic intermediates (cleavage complexes). The aim of the present study was to define the structural relationship between the site(s) of covalently bound intercalating agents, whose solution conformations in DNA are known, and the site(s) of top1 cleavage. Two diastereomeric pairs of oligonucleotide 22-mers, derived from a sequence used to determine the crystal structure of top1-DNA complexes, were synthesized. One pair contained either a trans-opened 10R- or 10S-benzo[a]pyrene 7, 8-diol 9,10-epoxide adduct at the N(6)-amino group of a central 2'-deoxyadenosine residue in the scissile strand, and the other pair contained the same two adducts in the nonscissile strand. These adducts were derived from the (+)-(7R,8S,9S,10R)- and (-)-(7S,8R,9R, 10S)-7,8-diol 9,10-epoxides in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans. On the basis of analogy with known solution conformations of duplex oligonucleotides containing these adducts, we conclude that top1 cleavage complexes are trapped when the hydrocarbon adduct is intercalated between the base pairs flanking a preexisting top1 cleavage site, or between the base pairs immediately downstream (3' relative to the scissile strand) from this site. We propose a model with the +1 base rotated out of the duplex, and in which the intercalated adduct prevents religation of the corresponding nucleotide at the 5' end of the cleaved DNA. These results suggest mechanisms whereby intercalating agents interfere with the normal function of human top1.

New and highly efficient synthesis of cis- and trans-opened Benzo[a]pyrene 7,8-diol 9,10-epoxide adducts at the exocyclic N(2)-amino group of deoxyguanosine.

We describe a new and facile method for the synthesis of both cis- and trans-opened N(2)-deoxyguanosine (dG) adducts of (+/-)-7alpha, 8beta-dihydoxy-9beta,10beta-epoxy-7,8,9,10-tetra hydrobenzo[a]pyrene and (+/-)-7alpha,8beta-dihydoxy-9alpha,10alpha -epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene at C-10. The key step in our approach is the direct coupling of O(6)-allyl-3', 5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine with these epoxides followed by the separation of the mixtures of cis- and trans-diastereomers produced. Overall coupling yields ranged from 45 to 65%. Stereochemistry of addition of the N(2)-exocyclic amino group of dG (cis-trans, approximately 1:1) was assigned by NMR, and the absolute configuration of the dG adducts was unequivocally assigned by CD spectroscopy after separation of each individual diastereomer and cleavage of the allyl protecting group. A strong CD band at 279 nm in the O(6)-protected adduct was found to be diagnostic for configuration at C-10, with a negative band correlating with 10R configuration. The synthetic methodology described allows easy access to cis- and trans-opened N(2)-dG adducts which are valuable building blocks for the synthesis of adduct-containing oligonucleotides for physical and biochemical studies.

Incorporation of 4-thiothymidine into DNA by the Klenow fragment and HIV-1 reverse transcriptase.

The 5'-triphosphate of 4-thiothymidine (4S-TTP) is an excellent substrate for the Klenow fragment of Escherichia coli DNA polymerase 1 and HIV-1 reverse transcriptase with values of k(cat)/Km within a factor of approximately 3 of those for TTP. A large UV change (deltaepsilon= -9770 M(-1)cm(-1) at 340 nm) associated with incorporation of 4S-TMP into nucleic acid duplexes makes possible a rapid, continuous spectrophotometric assay of the reaction progress.

Factors determining mutagenic potential for individual cis and trans opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts.

Four adducts that would result from trans opening at C-1 of benzo[c]phenanthrene 3,4-diol 1,2-epoxide (B[c]PhDE) isomers (i.e., DE-1 enantiomers, where the epoxide oxygen and benzylic hydroxyl group are cis, and DE-2 enantiomers, where they are trans) by the N(6)-amino group of dAdo, together with the two cis opened N(6)-dAdo adducts of B[c]PhDE-1, were incorporated into two oligonucleotides at the underlined site in 5'-TTTAGAGTCTGCTCCC [context I(A)] and 5'-CAGATTTAGAGTCTGC [context II(A)]. After ligation of these, and the corresponding unsubstituted oligonucleotides, into single-stranded M13mp7L2 bacteriophage and transfection into SOS-induced Escherichia coli SMH77, base substitution mutations induced by the different B[c]PhDE-dAdo adducts were determined. These findings were compared with data [Pontén et al. (1999) Biochemistry 38, 1144-1152] for cis opened B[c]PhDE-2-dAdo adducts in the same sequence contexts. In most cases, adducts with S absolute configuration at the site of attachment of the nucleoside to the hydrocarbon had higher mutation frequencies (1.9-56.5%) than the corresponding adducts with R configuration (0.05-5.6%). For adducts derived from B[c]PhDE-1, the predominant mutations were A-->T transversions in context I(A) and A-->G transitions for most of these adducts in context II(A). For adducts derived from B[c]PhDE-2, A-->T base substitutions predominated for most of the trans adducts, but A-->G mutations were favored by the cis adduct with S configuration in either context. Thus, the structural feature that most dramatically affected mutagenic activity was the configuration of the carbon at the attachment point, with S configuration mostly being associated with greater mutagenicity than the R configuration. However, other structural variations and sequence context also affected mutagenicity, indicating that a combination of structure and context effects define mutagenicity.

Benzo[a]pyrene diol epoxide adducts in DNA are potent suppressors of a normal topoisomerase I cleavage site and powerful inducers of other topoisomerase I cleavages.

The catalytic intermediates of DNA topoisomerase I (top1) are cleavage complexes that can relax DNA supercoiling (intramolecular reaction) or mediate recombinations (intermolecular religation). We report here that DNA adducts formed from benzo[a]pyrene bay-region diol epoxides can markedly affect top1 activity. Four oligonucleotide 22-mers of the same sequence were synthesized, each of which contained a stereoisomerically unique benzo[a]pyrene 7, 8-diol 9,10-epoxide adduct at the 2-amino group of a central 2'-deoxyguanosine residue. These four adducts correspond to either cis or trans opening at C-10 of the (+)-(7R, 8S, 9S, 10R)- or (-)-(7S, 8R, 9R, 10S)-7,8-diol 9,10-epoxides. Their solution conformations in duplex DNA (intercalated and minor-groove bound for the cis and trans opened adducts respectively) can be deduced from previous NMR studies. All four adducts completely suppress top1 cleavage activity at the alkylation site and induce the formation of new top1cleavage complexes on both strands of the DNA 3-6 bases away from the alkylation site. The trans opened adduct from the highly carcinogenic (+)-diol epoxide is the most active in inducing top1 cleavage independently of camptothecin, demonstrating that minor groove alkylation can efficiently poison top1. We also found that this isomer of the diol epoxide induces the formation of top1-DNA complexes in mammalian cells, which suggests a possible relationship between induction of top1 cleavage complexes and carcinogenic activity of benzo[a]pyrene diol epoxides.