Search PubMed⌕ Search

Biomedical subjects

T M Harris

Publications and source records attributed to T M Harris.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1998

An intercalated and thermally stable FAPY adduct of aflatoxin B1 in a DNA duplex: structural refinement from 1H NMR.

The structure of a formamidopyrimidine (FAPY) adduct arising from imidazole ring opening of the initially formed trans-8, 9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct under basic conditions and positioned in the 5'-d(CTATFAPYGATTCA)-3'*5'-d(TGAATCATAG)-3' oligodeoxynucleotide was determined. The FAPY adduct may be a major progenitor of aflatoxin B1-induced mutations in DNA. The freshly prepared sample showed biphasic melting, with transitions at 28 and 56 degreesC. NMR initially showed multiple subspectra. Over a period of several days at 4 degreesC, the sample converted to a single species with a Tm of 56 degreesC, 15 degrees C greater than the unmodified duplex. The deoxyribose was in the beta configuration about the anomeric carbon, evidenced by NOEs between FAPYG5 H3', H2', H2", and H1'. FAPY formation resulted in the loss of the guanine H8 proton, and the introduction of the formyl proton, which showed NOEs to FAPYG5 H1' and A6 N6Ha. A total of 31 NOEs from AFB1 to DNA protons were observed, mostly to the 5'-neighboring base, T4 in the modified strand. Sequential NOEs were interrupted between T4 and FAPYG5 in the modified strand, between C16 and A17 in the complementary strand, and between T4 N3H and FAPYG5 N1H. An NOE between FAPYG5 N1H and C16 N4H showed intact hydrogen bonding at FAPYG5*C16. Upfield chemical shifts were observed for T4 H6 and A17 H8. Molecular dynamics calculations converged with pairwise rmsd differences of <0.9 A. The sixth root residual was 8.7 x 10(-2). The AFB1 moiety intercalated from the major groove between FAPYG5 and T4*A17, and stacked with T4 and FAPYG5 and partially stacked with A17. The base step between T4*A17 and FAPYG5*C16 was increased from 3.4 to 7 A. The duplex unwound by about 15 degrees. The FAPY formyl group was positioned to form a hydrogen bond with A6 N6Ha. Strong stacking involving the AFB1 moiety, and this hydrogen bond explains the thermal stabilization of four base pairs by this adduct, and may be a significant factor in its processing.

Aflatoxin B1↗
PubMed1998

Reactivation of the maternally imprinted IGF2 allele in TGFalpha induced hepatocellular carcinomas in mice.

The Insulin like growth factor 2 (IGF2) gene is expressed in several types of tumors in humans and mice and has been implicated as an important growth factor in tumor progression. IGF2 expression in the TGF alpha transgenic mice was analysed in liver and tumors from animals which also contained one or two functional IGF2 alleles. In a two by two mating experiment using transgenic mice containing either a TGF alpha transgene or a IGF2 gene knockout, we have investigated whether IGF2 imprinting is reversed during hepatocarcinogenesis and the consequences of IGF2 expression for tumor growth. We observed that: (1) 100% of the hepatocellular carcinomas expressed IGF2 (2) the normally imprinted maternal allele is active in the tumors in which the paternal allele is knocked out and (3) all three of the murine IGF2 promoters upstream of the reactivated maternal alleles are transcriptionally active in tumors. We also observed that the total tumor burden of animals with two wild type IGF-2 alleles (paternal and maternal) was the same as the tumor burden in animals which contained only a single reactivated maternal allele. The 100% incidence of reactivation of the imprinted maternal allele suggests that IGF2 expression is selected during murine hepatocarcinogenesis and can substitute for the paternal allele when it is inactivated.

Alleles↗
PubMed1998

Styrene oxide adducts in an oligodeoxynucleotide containing the human N-ras codon 12: minor groove structures of the R(12,1)- and S(12,1)-alpha-(N2-guanyl) stereoisomers determined by 1H nuclear magnetic resonance.

The R- and S-alpha-(N2-guanyl)styrene oxide (SO) adducts at X5 in d(G1G2C3A4X5G6T7G8G9T10G11).d(C12A13C14C15A16C17C18T19G20C21C22 ), encompassing codon 12 of the human N-ras protooncogene (underlined), were examined using 1H NMR spectroscopy. These were the R(12,1) and S(12,1) adducts, indicating the location of the R or S adduct at the first position of codon 12. These differed from the R- and S(12, 2)-alpha-SO adducts [Zegar, I. S., Setayesh, F. R., DeCorte, B. L., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 4334-4348] in that the base pair 5' to the lesion was changed from G.C to A.T, while the base pair 3' to the lesion was changed from T.A to G.C. Comparison of the R- and S(12,1) adducts with the R- and S(12,2) adducts allowed the effects of flanking bases on the conformations of the alpha-SO adducts to be examined. This change in flanking base affected the R-SO lesion. The R(12,1) adduct structure was disordered at the adduct site, and a refined structure could not be obtained. NOE and chemical shift data suggested that the styrenyl moiety was oriented in the minor groove and in the 3'-direction from the site of adduction. In contrast, this change in flanking base did not affect the S-SO lesion. The S(12,1) adduct yielded a refined structure, with the styrenyl moiety edgewise in the minor groove and oriented in the 5'-direction relative to the site of adduction. A total of 232 interproton distances, including 13 styrene-DNA distances, were obtained. A total of 12 NOE-restrained molecular dynamics calculations converged with pairwise root-mean-square deviation of 1.10 A. The sixth-root residual index between calculated and experimental NOE intensities was 8.0 x 10(-)2 A. The styrene aromatic protons appeared as three resonances, suggesting rapid rotation. The possibility of a hydrogen bond between the styrene hydroxyl and C18 O2 in the S(12,1) adduct could not be confirmed. This work illustrates the dual roles of stereochemistry and sequence in modulating the properties of guanine N2 alpha-SO adducts.

Codon↗
PubMed1998

Metabolic labeling of woodchuck hepatitis B virus X protein in naturally infected hepatocytes reveals a bimodal half-life and association with the nuclear framework.

In order to identify potential sites of hepadnavirus X protein action, we have investigated the subcellular distribution and the stability of woodchuck hepatitis virus (WHV) X protein (WHx) in primary hepatocytes isolated from woodchucks with persistent WHV infection. In vivo cell labeling and cell fractionation studies showed that the majority of WHx is a soluble cytoplasmic protein while a minor part of newly synthesized WHx is associated with a nuclear framework fraction (20%) and with cytoskeletal components (5 to 10%). Pulse-chase experiments revealed that cytoplasmic WHx has a short half-life and decays with bimodal kinetics (approximately 20 min and 3 h). The rates of association and turnover of nucleus-associated WHx suggest that compartmentalization may be responsible for the bimodal turnover observed in the cytoplasm.

Animals↗
PubMed1997

Lack of correlation between in vitro and in vivo replication of precisely defined benz-a-anthracene adducted DNAs.

Like other polycyclic aromatic hydrocarbons, certain metabolites of benz[a]anthracene have been implicated as potent carcinogens. These effects are thought to be caused by the covalent binding of these species to nucleophilic groups on the bases of DNA. To address the molecular mechanisms by which these molecules induce mutations, this study employed oligonucleotides containing four site-specific N6 adenine-benz[a]anthracene diol epoxide adducts. Using a prokaryotic in vivo replication system, we have shown that both non-bay region anti-trans-benz[a]anthracene adducts are essentially nonmutagenic. In contrast, the bay region anti-trans-benz[a]anthracene lesions do induce point mutations at the adduct site. The mutagenic frequency of these bay region lesions is dependent on the stereochemistry about the adduct-forming bond, as well as the strain of Escherichia coli in which they are replicated. The ability of the bacterial replication machinery to bypass the lesions does not correlate with the differences observed in their mutagenesis. While both non-bay region adducts are readily bypassed in vivo, the bay region adducts are both blocking to approximately the same degree. In vitro studies of the interactions of E. coli DNA polymerase III with these adducts have also been undertaken to further dissect the relationship between adduct structure and biological activity.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1997

Differential tolerance to DNA polymerization by HIV-1 reverse transcriptase on N6 adenine C10R and C10S benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide-adducted templates.

To determine the effect of various stereoisomers of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE) on translesion bypass by human immunodeficiency virus-1 reverse transcriptase and its alpha-helix H mutants, six 33-mer templates were constructed bearing site- and stereospecific adducts. This in vitro model system was chosen to understand the structure-function relationships between the polymerase and damaged DNA during replication. Comparison of the replication pattern between wild type human immunodeficiency virus-1 reverse transcriptase and its mutants, using primers which were 3' to the lesion, revealed essentially similar patterns. While these primers terminated with all three of the C10R and two of the C10S BPDE-adducted templates 1 base 5' and 1 base 3' to the damaged site respectively, (+)-anti-trans-(C10S) BPDE-adducted DNA alone permitted the formation of full-length products. Utilization of a primer with its 3'-hydroxyl 1 base beyond the lesion resulted in full-length products with all the C10S BPDE-adducted templates and the (-)-syn-trans-(C10R)-BPDE-adducted template, following replication with either the wild type or mutant enzymes. However, the other two C10R BPDE-adducted templates failed to allow any primer extension, even with the wild type enzyme. Although T.P depletion studies further confirmed the differential primer extension abilities using the C10R and C10S adducted templates, their binding affinities were similar, yet distinct from the unadducted template.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1997

Characterization of DNA adducts formed by cyclopenta[cd]pyrene epoxide.

Cyclopenta[cd]pyrene (CPP) is a ubiquitous environmental pollutant whose 3,4-epoxide (CPPE) is generally regarded as its ultimate carcinogenic metabolite. The present study was undertaken to determine the structures of major DNA adducts formed by CPPE in vitro. Incorporation of specific radiolabeled bases into calf thymus DNA prior to reaction with CPPE demonstrated that the major adducts were formed by guanine, while minor adducts were formed by adenine and cytosine. Unmodified DNA was reacted with [3H]CPPE and the deoxynucleoside adducts obtained were compared chromatographically with the products obtained by reaction of CPPE with 2'-deoxyguanosine (dGuo). Two dGuo adducts from the latter reaction were identified by 1H-NMR, fast atom bombardment mass spectrometry, and circular dichroism as diastereoisomers of cis-3-(deoxyguanosin-N2-yl)-4-hydroxy-3,4-dihydroCPP. Other products that may have included the isomeric trans-N2-dGuo adduct were formed in the reaction. The major adduct fraction in the DNA digest, accounting for over 70% of the total, was chromatographically indistinguishable from the two cis dGuo-N2 adducts. A second DNA adduct fraction was observed, which appeared also to be formed by reaction with guanine as indicated by experiments in which DNA containing [3H]guanine was reacted with unlabeled CPPE. The results confirm that guanine is the major target in DNA for reaction with CPPE and are the first proof of structure for a CPPE-deoxynucleoside adduct.

Animals↗