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T M Harris

Publications and source records attributed to T M Harris.

At least 19 recordsLinked to original sources
PubMed2000

Evaluation of the mutagenic potential of the principal DNA adduct of acrolein.

Acrolein is produced extensively in the environment by incomplete combustion of organic materials, and it arises endogenously in humans as a metabolic by-product. Acrolein reacts with DNA at guanine residues to form the exocyclic adduct, 8-hydroxypropanodeoxyguanosine (HOPdG). Acrolein is mutagenic, and a correlation exists between HOPdG levels in Salmonella typhimurium treated with acrolein and a resultant increase in mutation frequency. Site-specifically modified oligonucleotides were used to explore the mutagenic potential of HOPdG in Escherichia coli strains that were either wild-type for repair or deficient in nucleotide excision repair or base excision repair. Oligonucleotides modified with HOPdG were inserted into double-stranded bacteriophage vectors using the gapped-duplex method or into single-stranded bacteriophage vectors and transformed into SOS-induced E. coli strains. Progeny phage were analyzed by oligonucleotide hybridization to establish the mutation frequency and the spectrum of mutations produced by HOPdG. The correct base, dCMP, was incorporated opposite HOPdG in all circumstances tested. In contrast, in vitro lesion bypass studies showed that HOPdG causes misincorporation opposite the modified base and is a block to replication. The combination of these studies showed that HOPdG is not miscoding in vivo at the level of sensitivity of these site-specific mutagenesis assays.

Acrolein↗
PubMed2000

Butadiene-induced intrastrand DNA cross-links: a possible role in deletion mutagenesis.

To initiate studies designed to identify the mutagenic spectrum associated with butadiene diepoxide-induced N(2)-N(2) guanine intrastrand cross-links, site specifically adducted oligodeoxynucleotides were synthesized in which the adducted bases were centrally located within the context of the human ras 12 codon. The two stereospecifically modified DNAs and the corresponding unmodified DNA were ligated into a single-stranded M13mp7L2 vector and transfected into Escherichia coli. Both stereoisomeric forms (R, R and S,S) of the DNA cross-links resulted in very severely decreased plaque-forming ability, along with an increased mutagenic frequency for both single base substitutions and deletions compared with unadducted DNAs, with the S,S stereoisomer being the most mutagenic. Consistent with decreased plaque formation, in vitro replication of DNA templates containing the cross-links by the three major E. coli polymerases revealed replication blockage by both stereoisomeric forms of the cross-links. The same DNAs that were used for replication studies were also assembled into duplex DNAs and tested as substrates for the initiation of nucleotide excision repair by the E. coli UvrABC complex. UvrABC incised linear substrates containing these intrastrand cross-links with low efficiency, suggesting that these lesions may be inefficiently repaired by the nucleotide excision repair system.

Adenosine Triphosphatases↗
PubMed2000

Sequence- and stereospecific conformational rearrangement of styrene oxide adducts located at A x C mismatched base pairs.

The solution structures of R- and S-alpha-(N(6)-adenyl)-styrene oxide adducts mismatched with cytosine at position X(7) in d(CGGACAXGAAG) x d(CTTCCTGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, were determined. These were the R- and S(61,3)C adducts. The structures for these mismatched adducts differed from the sequence isomeric R- and S(61,2)C adducts [Painter, S. L., Zegar, I. S., Tamura, P. J., Bluhm, S., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 8635-8646]. The results reveal that the structural consequences of cytosine mispairing opposite the R- and S-alpha-SO adducts differ as a function of DNA sequence. The thermodynamic stability of both the R- and S(61,3)C mismatched adducts was dependent upon pH. At neutral pH, the R- and S(61,3)C adducts exhibited significant structural perturbation and had lower T(m) values, as compared to the R- and S(61,2)C adducts. In both instances, this was attributed to reorientation about the C6-N(6) bond, such that the N(6)H proton faced away from the Watson-Crick face of the purine base and into the major groove. The conformation about the N(6)-C(alpha)-C(beta)-O torsion angle was predicted from rMD calculations to be stabilized by a N/O gauche-type interaction between the styrenyl hydroxyl moiety and adenine N(6) at the lesion site. For the R(61,3)C adduct, the styrenyl moiety remained oriented in the major groove and faced in the 3'-direction. In the properly base-paired R(61,3) adduct, it had faced in the 5' direction. For the S(61,3)C adduct, the styrene ring was inserted into the duplex, approximately perpendicular to the helical axis of the DNA. It faced in the 5'-direction. In the properly base-paired S(61,3) adduct, it had faced in the 3'-direction. The results were correlated with site-specific mutagenesis experiments in vivo. The latter revealed that the R- and S(61,3)-alpha-styrene oxide adducts were nonmutagenic. This may be a consequence of the greater structural perturbation associated with formation of the cytosine mismatch at neutral pH for the R- and S(61,3) adducts as compared to the S(61,2) adduct that exhibited low levels of A --> G mutations.

Adenine↗
PubMed2000

Laryngeal manipulation.

This article presents the authors' philosophy regarding the use of physical manipulation of the larynx and the neck in patients presenting with voice disorders from the context of the anatomy and physiology of the larynx. The biomechanics of the laryngeal structures are reviewed. Potential indications for manipulation are discussed. The examination of the larynx and perilaryngeal structures is presented from a mechanical standpoint. Some basic tenets in laryngeal manipulation, including potential risks and contraindications, are offered.

Humans↗
PubMed2000

Studies of the mechanisms of adduction of 2'-deoxyadenosine with styrene oxide and polycyclic aromatic hydrocarbon dihydrodiol epoxides.

The mechanism of adduction of 2'-deoxyadenosine by styrene oxide and polycyclic aromatic hydrocarbon dihydrodiol epoxides has been explored using (15)N(6)-labeled adenine nucleosides. The extent of reaction at N1 versus N(6) was evaluated by (1)H NMR of the N(6) adducts after allowing Dimroth rearrangement to occur. Products arising from attack at N1 followed by Dimroth rearrangement exhibited a small two-bond (1)H-(15)N coupling constant (N1-H2 J approximately 13 Hz); products from direct attack exhibited a much larger one-bond (1)H-(15)N coupling constant (J approximately 90 Hz). In the case of styrene oxide, all of the N(6) beta adduct arose by initial attack at N1, whereas the majority (70-80%) of the N(6) alpha adducts came from direct attack. The styrene oxide reaction was also studied with a self-complementary oligodeoxynucleotide (24-mer) containing nine (15)N(6)-labeled adenine residues. NMR examination of the N(6) alpha- and beta-styrene oxide adducts isolated after enzymatic degradation of the 24-mer gave very similar results, indicating that N1 attack can occur readily even with a duplexed oligonucleotide. With the PAH dihydrodiol epoxides, only naphthalene dihydrodiol epoxide exhibited significant initial reaction at N1 (50%). No detectable rearranged product was seen in reactions with benzo[a]pyrene dihydrodiol epoxide or non-bay or bay region benz[a]anthracene dihydrodiol epoxide; interestingly, a small amount of N1 attack (5-7%) was seen in the case of benzo[c]phenanthrene dihydrodiol epoxide. It appears that initial attack at N1 is only a significant reaction pathway for epoxides attached to a single aromatic ring.

Base Sequence↗
PubMed2000

Mutagenic potential of guanine N2 adducts of butadiene mono- and diolepoxide.

To explore the role of guanine N(2) adducts of stereoisomeric butadiene metabolites in butadiene-induced mutagenesis, 11-mer deoxyoligonucleotides were prepared containing adducts of (R)- and (S)-monoepoxide and (R,R)- and (S,S)-diolepoxide. These adducted oligonucleotides were utilized in both in vivo and in vitro experiments designed to examine the mutagenic potency of each and their replication by Escherichia coli polymerases. Each of the four adducted deoxyoligonucleotides was ligated into a single-stranded M13mp7L2 vector and transfected into E. coli. The resulting plaques were screened for misincorporation at position 2 of the N-ras 12 codon. Although the mutagenic frequencies were low, different relative mutagenicities of the various stereoisomers were discernible. In addition, the biological effects of each adduct on the three major E. coli polymerases were determined via primer extension assays. The adducted 11-mers were ligated into a 60-mer linear DNA molecule to provide a sufficiently long template for primer elongation. All four guanine adducts were determined to be blocking to each of the three polymerases via primer extension assays.

Base Sequence↗
PubMed2000

Altered electrophoretic migration of polycyclic aromatic hydrocarbon and styrene oxide adducts at adenine N(6) correlates with adduct-induced structural disorder.

Site-specific bay region benzo[a]pyrene (7R,8S,9R,10S)-N(6)-[10-(7,8, 9,10-tetrahydro-7,8,9-trihydroxybenzo[a]pyrenyl)]-2'-deoxyadeno syl, (7S,8R,9S,10R)-N(6)-[10-(7,8,9,10-tetrahydro-7,8, 9-trihydroxybenzo[a]pyrenyl)]-2'-deoxyadenosyl, (7S,8R,9R, 10S)-N(6)-[10-(7,8,9,10-tetrahydro-7,8, 9-trihydroxybenzo[a]pyrenyl)]-2'-deoxyadenosyl, and (7R,8S,9S, 10R)-N(6)-[10-(7,8,9,10-tetrahydro-7,8, 9-trihydroxybenzo[a]pyrenyl)]-2'-deoxyadenosyl adducts, bay region benz[a]anthracene (1R,2S,3R,4S)-N(6)-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl and (1S,2R,3S, 4R)-N(6)-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adducts, non-bay region benz[a]anthracenyl (8S,9R,10S,11R)-N(6)-[11-(8,9,10, 11-tetrahydro-8,9,10-trihydroxybenz[a]anthracenyl)]-2'-de oxyadenosyl and (8R,9S,10R,11S)-N(6)-[11-(8,9,10,11-tetrahydro-8,9, 10-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adducts, and the R- and S-adducts of styrene oxide were located in the ras61 oligodeoxynucleotide and examined with respect to electrophoretic mobility. The results were compared to NMR structural data, and to site-specific mutagenesis data and in vitro DNA replication assays for the same adducts. There was a correlation between adducts having lower electrophoretic mobility and greater disorder at the adduct site as monitored by NMR. The disorder combined with the lower electrophoretic mobilities suggested that these adducts induced flexible hinge joints in the DNA rather than static bending. Usually, these were adenine N(6) adducts having S-stereochemistry at the benzylic carbon. The results also revealed a possible role for the bay region ring in stabilizing adenyl N(6) benz[a]anthracene adducts with respect to hinging at the adduct site. On the other hand, there was not a simple relationship between altered electrophoretic mobility and mutagenesis or DNA replication.

Adenine↗
PubMed2000

Synthesis of nucleosides and oligonucleotides containing adducts of acrolein and vinyl chloride.

Vinyl chloride and acrolein are important industrial chemicals. Both form DNA adducts, vinyl chloride after enzymatic oxidation to chlorooxirane and acrolein by direct reaction. Reaction at the N(2) position of guanine is a major pathway. The resulting 2-oxoethyl and 3-oxopropyl adducts cyclize spontaneously to hydroxyethano and hydroxypropano derivatives, respectively. The two cyclic adducts have been detected in DNA exposed to these mutagens. A new method has been developed for the synthesis of deoxyguanosine adducts of chlorooxirane and acrolein, as well as oligonucleotides containing these adducts. Reaction of O(6)-[(trimethylsilyl)ethyl]-2-fluoro-2'-deoxyinosine with the appropriate aminodiol followed by oxidative cleavage of the diol with NaIO(4) gave the adducts in excellent yields. Reaction of oligonucleotides containing the halonucleoside with the aminodiols followed by NaIO(4) efficiently created the nucleosides in the oligonucleotides. Deoxyadenosine adducts were created similarly using 6-chloropurine 9-(2'-deoxyriboside).

Acrolein↗
PubMed2000

DNA interstrand cross-links induce futile repair synthesis in mammalian cell extracts.

DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5' to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3'-to-5' exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.

Animals↗
PubMed2000

Mutagenic potential of adenine N(6) adducts of monoepoxide and diolepoxide derivatives of butadiene.

To determine the biological effects of specific DNA adducts resulting from the interaction of 1,3-butadiene metabolites with DNA, deoxyoligonucleotides have been synthesized with four different adducts at the N(6) position of adenine, centrally located within the human N-ras codon 61. The adducts are those arising from adduction by either the R or S stereoisomer of the monoepoxide (BDO) or the (R,R) or (S,S) isomer of the diolepoxide (BDE). The diolepoxide can arise from partial hydrolysis of the diepoxide (BDO(2)) or from epoxidation of hydrolyzed monoepoxide. These adducted oligonucleotides were used in in vivo and in vitro assays designed both to determine their mutagenic potency and to examine specific interactions with Escherichia coli polymerases. Each adducted oligonucleotide was ligated into a single-stranded vector M13mp7L2 that was subsequently used to transfect E. coli. The resulting mutagenic spectrum for these modified DNAs was stereoisomer specific. Both monoepoxide lesions were nonmutagenic, but the mutagenic spectra for the modified DNAs containing BDE adducts were stereoisomer specific. The mutations generated by adducts of the R,R enantiomer of the diolepoxide were exclusively A --> G, whereas adducts of the S,S enantiomer of the diolepoxide yielded exclusively A --> C mutations. None of the four modifications resulted in significant blocks to in vivo phage replication, as evidenced by no decrease in plaque-forming ability. Consistent with these data, when each of three purified E. coli polymerases was used to replicate DNAs containing these adducted deoxyoligonucleotides, the individual polymerases appeared to be virtually unaffected, such that all lesions were readily bypassed. Whereas previous animal model studies identified the mutagenic spectrum related to butadiene exposure, these studies begin to establish the specific lesions responsible for mutagenesis. This is the first report of stereoselectivity related to butadiene-induced mutagenesis.

Adenine↗
PubMed1999

Intercalation of the (1S,2R,3S,4R)-N6-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in an oligodeoxynucleotide containing the human N-ras codon 61 sequence.

The (1S,2R,3S,4R)-N(6)-[1-(1,2,3,4-tetrahydro-2,3, 4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, results from trans opening of (1R,2S,3S,4R)-1,2-epoxy-1,2,3, 4-tetrahydrobenz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Two conformations of this adduct exist, in slow exchange on the NMR time scale. A structure for the major conformation, which represents approximately 80% of the population, is presented. In this conformation, an anti glycosidic torsion angle is observed for all nucleotides, including S,R,S,RA6. The refined structure is a right-handed duplex, with the benz[a]anthracene moiety intercalated on the 3'-face of the modified base pair, from the major groove. It is located between S,R,S,RA6.T17 and A7.T16. Intercalation is on the opposite face of the modified S,R,S,RA6.T17 base pair as compared to the (1R,2S,3R,4S)-N6-[1-(1,2,3,4-tetrahydro-2, 3,4-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct, which intercalated 5' to the modified R,S,R,SA6.T17 base pair [Li, Z. , Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981]. The spectroscopic data do not allow refinement of the minor conformation, but suggest that the adenyl moiety in the modified nucleoti111S,R, S,RA6 adopts a syn glycosidic torsion angle. Thus, the minor conformation may create greater distortion of the DNA duplex. The results are discussed in the context of site-specific mutagenesis studies which reveal that the S,R,S,RA6 lesion is less mutagenic than the R,S,R,SA6 lesion.

Adenine↗
PubMed1999

Role of a polycyclic aromatic hydrocarbon bay region ring in modulating DNA adduct structure: the non-bay region (8S,9R,10S, 11R)-N(6)-[11-(8,9,10,11-tetrahydro-8,9, 10-trihydroxybenz[a]anthracenyl)]-2'-deoxyadenosyl adduct in codon 61 of the human N-ras protooncogene.

The structure of the non-bay region (8S,9R,10S,11R)-N(6)-[11-(8,9,10, 11-tetrahydro-8,9,10-trihydroxybenz[a]anthracenyl)]-2'-de oxyadenosyl adduct at X(6) of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, was determined. Molecular dynamics simulations were restrained by 475 NOEs from (1)H NMR. The benz[a]anthracene moiety intercalated above the 5'-face of the modified base pair and from the major groove. The duplex suffered distortion at and immediately adjacent to the adduct site. This was evidenced by the disruption of the Watson-Crick base pairing for X(6) x T(17) and A(7) x T(16) and the increased rise of 7.7 A between base pairs C(5) x G(18) and X(6) x T(17). Increased disorder was observed as excess line width of proton resonances near the lesion site. Comparison with the bay region benzo[a]pyrene [Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212-6224] and bay region benz[a]anthracene [Li, Z., Mao, H., Kim, H.-Y., Tamura, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1999) Biochemistry 38, 2969-2981] adducts with the corresponding stereochemistry and at the same site shows that this non-bay region benz[a]anthracene lesion assumes different base pair geometry, in addition to exhibiting greater disorder. These differences are attributed to the loss of the bay region ring. The results suggest the bay region ring contributes to base stacking interactions at the lesion site. These structural differences between the non-bay and bay region lesions are correlated with site-specific mutagenesis data. The bay region benzo[a]pyrene and bay region benz[a]anthracene adducts were poorly replicated in vivo, and induced A --> G mutations. In contrast, the non-bay region benz[a]anthracene adduct was easily bypassed in vivo and was nonmutagenic.

Bay-Region, Polycyclic Aromatic Hydrocarbon↗
PubMed1999

Influence of the R(61,2)- and S(61,2)-alpha-(N6-adenyl)styrene oxide adducts on the A.C mismatched base pair in an oligodeoxynucleotide containing the human N-ras codon 61.

Conformational studies of R- and S-alpha-(N6-adenyl)styrene oxide adducts mismatched with deoxycytosine at position X6 in d(CGGACXAGAAG).d(CTTCTCGTCCG), incorporating codons 60, 61 (underlined), and 62 of the human N-ras protooncogene, are described. These were the R- and S(61,2)C adducts. The S(61,2)C adduct afforded a stable solution structure, while the R(61,2)C adduct resulted in a disordered structure. Distance restraints for the S(61, 2)C adduct were calculated from NOE data using relaxation matrix analysis. These were incorporated as effective potentials into the total energy equation. The structures were refined using restrained molecular dynamics calculations which incorporated a simulated annealing protocol. The accuracy of the emergent structures was evaluated by complete relaxation matrix methods. The structures refined to an average rms difference of 1.07 A, determined by pairwise analysis. The experimentally determined structure was compared to NOE intensity data using complete relaxation matrix back-calculations, yielding an R1x value of 11.2 x 10(-)2. The phenyl ring of the styrene in the S(61,2)C adduct was in the major groove and remained oriented in the 3'-direction as observed for the corresponding S(61,2) adduct paired with thymine [Feng, B., Zhou, L., Pasarelli, M., Harris, C. M., Harris, T. M., and Stone, M. P. (1995) Biochemistry 34, 14021-14036]. A shift of the modified adenine toward the minor groove resulted in the styrenyl ring stacking with nucleotide C5 on the 5'-side of the lesion, which shifted toward the major groove. Unlike the unmodified A.C mismatch, neither the S(61,2)C nor the R(61,2)C adduct formed protonated wobble A.C hydrogen bonds. This suggests that protonated wobble A.C pairing need not be prerequisite to low levels of alpha-SO-induced A --> G mutations. The shift of the modified adenine toward the minor groove in the S(61,2)C structure may play a more important role in the genesis of A --> G mutations. The disordered structure of the R(61,2)C adduct provides a potential explanation as to why that adduct does not induce A --> G mutations.

Adenine↗
PubMed1999

Intercalation of the (-)-(1R,2S,3R, 4S)-N6-[1-benz[a]anthracenyl]-2'-deoxyadenosyl adduct in an oligodeoxynucleotide containing the human N-ras codon 61 sequence.

The solution structure of the (-)-(1R,2S,3R,4S)-N6-[1-(1,2,3, 4-tetrahydroxy-benz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61(italic), and 62 of the human N-ras protooncogene, was determined. This adduct results from the trans opening of 1S,2R,3R,4S-1, 2-epoxy-1,2,3,4-tetrahydro-benz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Molecular dynamics simulations were restrained by 509 NOEs from 1H NMR. The precision of the refined structures was monitored by pairwise root-mean-square deviations which were <1.2 A; accuracy was measured by complete relaxation matrix calculations, which yielded a sixth root R factor of 9.1 x 10(-)2 at 250 ms. The refined structure was a right-handed duplex, in which the benz[a]anthracene moiety intercalated from the major groove between C5.G18 and R,S,R,SA6.T17. In this orientation, the saturated ring of BA was oriented in the major groove of the duplex, with the aromatic rings inserted into the duplex such that the terminal ring of BA threaded the duplex and faced toward the minor groove direction. The duplex suffered localized distortion at and immediately adjacent to the adduct site, evidenced by the increased rise of 8.8 A as compared to the value of 3.5 A normally observed for B-DNA between base pairs C5.G18 and R,S,R,SA6.T17. These two base pairs also buckled in opposite directions away from the intercalated BA moiety. The refined structure was similar to the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9, 10)-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct of corresponding stereochemistry at X6 of the same oligodeoxynucleotide [Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212-6224]. Both adducts intercalated toward the 5'-direction from the site of adduction. The similarities in solution structures were reflected in similar biological responses, when repair-deficient AB2480 Escherichia coli were transformed with M13mp7L2 DNA site-specifically modified with these two adducts.

Adenine↗
PubMed1999

Utilization of tetrabutylammonium triphenyldifluorosilicate (TBAT) in the synthesis of 6-fluoropurine nucleosides.

Tetrabutylammonium triphenydifluorosilicate (TBAT) has been found to be a useful reagent for the conversion of 6-chloropurine nucleosides to 6-fluoropurine derivatives. The 6-chloropurine nucleosides were reacted with trimethylamine to form quaternary trimethylammonium salts which were treated in situ with TBAT in DMF to effect conversion to the 6-fluoro derivatives in yields of 59-72%.

Nucleosides↗
PubMed1998

Multiple conformations of an intercalated (-)-(7S,8R,9S, 10R)-N6-[10-(7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct in the N-ras codon 61 sequence.

The structure of the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9, 10-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct at A7 of 5'-d(CGGACAAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', derived from trans addition of the exocyclic N6-amino group of dA to (-)-(7S,8R,9R, 10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-DE2], was determined using molecular dynamics simulations restrained by 532 NOEs from 1H NMR. This was named the SRSR(61,3) adduct, derived from the N-rasprotooncogene at and adjacent to the nucleotides encoding amino acid 61 (underlined) of the p21 gene product. The solution structure of this adduct was best described as a mixture of two conformations in rapid equilibrium on the NMR time scale. The two populations differed in the pseudorotation angle of the sugar ring for the 5'-neighboring base A6, as determined from scalar coupling data. One population, estimated to be present at 53%, had the A6 deoxyribose in the C2'-endo conformation, while in the second conformation the A6 deoxyribose was in the C3'-endo conformation. NOEs between C5, A6, and SRSRA7 were either disrupted or weakened, as were those in the complementary strand between C15, T16, and T17. Major groove NOEs were observed between the benzo[a]pyrene aromatic protons, H1, H2, H3, H4, H5, and H6, and T16 CH3. Minor groove NOEs were observed between H1, H2, and H3 of benzo[a]pyrene and T16 H1' and H2' and T17 H1' and H2'. The benzo[a]pyrene protons H10, H11, and H12 showed NOEs to A6 H1', H2', and H2". The chemical shifts of the pyrenyl moiety were dispersed over a 1.9 ppm range. Upfield chemical shifts of 2.4 ppm for T16 N3H, 1.1 ppm for T17 N3H, 1.3 and 1.0 ppm for T16 H6 and CH3, 0.85 ppm for T16 H1', and 0.80 and 0.90 ppm for C15 H2' and H2" were observed. These observations were consistent with intercalation of the pyrenyl moiety toward the 5' direction of SRSRA7. The results were compared to the isomeric SRSR(61,2) adduct [I. S. Zegar, S. J. Kim, T. N. Johansen, P. J. Horton, C. M. Harris, T. M. Harris, and M. P. Stone (1996) Biochemistry 35, 6212-6224] and revealed the role of DNA sequence in modulating the conformation of this benzo[a]pyrene adduct.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1998

Highly mutagenic bypass synthesis by T7 RNA polymerase of site-specific benzo[a]pyrene diol epoxide-adducted template DNA.

We have previously developed an in vitro system that allows quantitative evaluation of the fidelity of transcription during synthesis on a natural template in the presence of all four nucleotides. Here, we have employed this system using a TAA ochre codon reversion assay to examine the fidelity of transcription by T7 RNA polymerase past an adenine residue adducted at the N6-position with (-)-anti-trans- or (+)-anti-trans-benzo[a]pyrene diol epoxide (BPDE). T7 RNAP was capable of transcribing past either BPDE isomer to generate full-length run-off transcripts. The extent of bypass was found to be 32% for the (-)-anti-trans-isomer and 18% for the (+)-anti-trans-isomer. Transcription past both adducts was highly mutagenic. The reversion frequency of bypass synthesis of the (-)-anti-trans-isomer was elevated 11,000-fold and that of the (+)-anti-trans-isomer 6000-fold, relative to the reversion frequency of transcription on unadducted template. Adenine was misinserted preferentially, followed by guanine, opposite the adenine adducted with either BPDE isomer. Although base substitution errors were by far the most frequent mutation on the adducted template, three- and six-base deletions were also observed. These results suggest that transcriptional errors, particularly with regard to damage bypass, may contribute to the mutational burden of the cell.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗