PubMed Health⌕ Search

Biomedical subjects

E L Loechler

Publications and source records attributed to E L Loechler.

At least 37 records · Page 2Linked to original sources
PubMed1995

Are base substitution and frameshift mutagenesis pathways interrelated? An analysis based upon studies of the frequencies and specificities of mutations induced by the (+)-anti diol epoxide of benzo[a]pyrene.

(+)-anti-B[a]PDE-induced mutagenesis is being investigated, including in a supF gene of the E. coli plasmid pUB3. Based upon various findings a working hypothesis was proposed that the major adduct of (+)-anti-B[a]PDE (formed at N2-Gua) is able to induce different base substitution mutations (e.g., GC-->TA vs. GC-->AT vs. GC-->CG) depending upon its conformation in DNA, which can be influenced by various factors, such as DNA sequence context. Frameshift mutations are also significant and are analyzed herein. In virtually all cases one of three possibilities is observed: (1) some treatments change frameshift and base substitution mutation frequency (MF) in a quantitatively parallel fashion; (2) other treatments, which change frameshift MF, can change base substitution MF in a quantitatively reciprocal fashion; finally, (3) there are treatments that do not change frameshift MF, and also do not change base substitution MF. (Changes can be brought about by SOS induction, differing DNA sequence context, or heating adducted pUB3 prior to transformation. Why different kinds of changes result in (1) vs. (2) vs. (3) is discussed.) Thus, base substitution and frameshift mutagenesis pathways appear to be coupled in some way, which is most easily rationalized if both pathways are interrelated. The simplest mechanism to rationalize this coupling is that a single (+)-anti-B[a]PDE adduct in a single conformation can be bypassed via either a frameshift or a base substitution pathway. The surprising implication is that--although different conformations are likely to be required to induce different base substitution mutations (e.g., GC-->TA vs. GC-->AT; see above)--a single conformation can give rise to either a base substitution or a frameshift mutation. Frameshift and base substitution pathways must eventually diverge, and it is proposed that this is controlled by factors such as DNA sequence context.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

AP sites are not significantly involved in mutagenesis by the (+)-anti diol epoxide of benzo[a]pyrene: the complexity of its mutagenic specificity is likely to arise from adduct conformational polymorphism.

In previous work, mutations induced by the (+)-anti diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] were scored in the supF gene of the Escherichia coli plasmid pUB3 [Rodriguez & Loechler (1993) Biochemistry 32, 1759]. pUB3 was reacted with (+)-anti-B[a]PDE and then either (1) transformed immediately into E. coli or (2) heated at 80 degrees C for 10 min prior to transformation. Heating only released a small fraction of adducts (approximately 5%) and did not significantly affect the mutagenic pattern at most sites in supF. However, at the major base substitution hotspot, G115, principally G-->T mutations (87%) were obtained prior to heating, while after heating, G-->T mutations decreased (45%) and G-->A (21%) and G-->C (33%) mutations became more prevalent. One model for this result is that prior to heating a heat-labile adduct at G115 causes one pattern of mutagenesis, but after heating the labile adduct is hydrolyzed to an apurinic site (AP site), which causes a second mutational pattern. To test this, a role for AP sites generated from labile adducts by heating at 80 degrees C for 10 min is investigated. It is shown that when plasmid pUB3 contains 22 (+)-anti-B[a]PDE adducts, 0.6% (or fewer) are converted to AP sites as determined in an assay based upon the action of an AP-endonuclease. In a separate line of investigation not involving (+)-anti-B[a]PDE adducts, mutation frequency (MF) per AP site is estimated. (In these experiments, AP sites were introduced into pUB3 by the classic procedure of heating at 70 degrees C/pH 5.0 to hydrolyze purines.(ABSTRACT TRUNCATED AT 250 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Evidence from in vitro replication that O6-methylguanine can adopt multiple conformations.

The effect of O6-methylguanine (m6G) on replication, in a partially double-stranded defined 25-base oligonucleotide, has been studied under nonlimiting conditions of unmodified dNTPs and over an extended time period, using the Klenow fragment of Escherichia coli DNA polymerase I. The sequence surrounding m6G has flanking cytosines (C-m6G-C), and the initial steady-state kinetics have been reported. When the primer was annealed so that the first base to be replicated was m6G, replication was virtually complete in approximately 5 min, although the reaction appears biphasic. When annealed with a primer where thymine or cytosine is paired opposite template m6G, about half the molecules were replicated in the first 15 sec, and no significant further replication was seen over a 1-hr period. When m6G was dealkylated by DNA-O6-methylguanine-methyltransferase, replication was rapid with no blockage. These data suggest that there can be two (or more) conformations of m6G. In these studies the term syn refers to conformers interfering with base-pairing, whereas anti refers to those allowing such base-pairing. Previous physical studies by others indicate that syn- and anti-conformers of the methyl group relative to the N1 of guanine are possible. Here molecular modeling/computational studies are described, suggesting that syn- and anti-m6G can be of similar energy in DNA, and, therefore, these two conformers may explain the two types of species observed during in vitro replication. An alternative explanation could be the possibility that the different species may manifest differential interactions of m6G with Klenow fragment. These results may provide a rationale for why m6G lesions in vivo have been reported to be lethal as well as mutagenic.

Base Sequence↗
PubMed1993

Mutagenesis by the (+)-anti-diol epoxide of benzo[a]pyrene: what controls mutagenic specificity?

Mutagenesis by (+)-anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide [(+)-anti-B[a]PDE], an important mutagenic/carcinogenic metabolite of benzo[a]pyrene (B[a]P), is being studied in order to understand the factors that influence mutagenesis both quantitatively and qualitatively. A new mutational system, which permits the selection of supF- mutations in an Escherichia coli plasmid, pUB3, was used. The work described herein is an extension of previous work, which involved plasmid adduction and then immediate transformation (Rodriguez & Loechler, 1993), and began with the observation that mutation frequency (MF) decreased approximately 2-fold when the (+)-anti-B[a]PDE-adducted plasmid pUB3 is either (1) frozen and then thawed prior to transformation or (2) heated at 80 degrees C for 10 min prior to transformation. Several results suggest that this decrease is not due to the loss of labile adducts. To begin to understand this phenomenon, the mutagenic spectra are compared for (+)-anti-B[a]PDE in supF for the unheated (187 mutants), the freeze/thawed (134 mutants), and the heated (254 mutants) samples. In general, freeze/thawing and heating cause a decrease in all classes of mutations. Considering substitution mutations at G.C base pairs, which predominate, the mutagenic specificity for the combined data sets is GC-->TA (57%), GC-->AT (23%), and GC-->CG (20%). This raises the question, how does (+)-anti-B[a]PDE generate this complex mutagenic specificity, which contrasts with the situation for, e.g., simple methylating agents? One factor is that mutagenic specificity at a particular guanine residue can be influenced by the base on its immediate 5'-side, most notably where mutations are virtually exclusively restricted to GC-->TA in 5'-TG-3' sequence contexts. One unexpected finding may provide additional insight. G115 in supF, which is the major hot spot for base-pairing mutagenesis, is the only site where the qualitative pattern of mutagenesis is significantly affected by heating the (+)-anti-B[a]PDE-adducted plasmid prior to transformation. Without heating, G115-->T mutations predominate, but following heating there is a statistically significant increase in the fraction of G115-->A and G115-->C mutations. The most likely model to explain this and other results is (1) a particular DNA adduct can adopt multiple conformations, (2) the conformation adopted by an adduct can be influenced by various factors, including DNA sequence context, as well as heating and freeze/thawing, and (3) each of these conformations can cause a different pattern of mutation.(ABSTRACT TRUNCATED AT 400 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Mutagenic specificity of the (+)anti-diol epoxide of dibenz[a,j]anthracene in the supF gene of an Escherichia coli plasmid.

This study was designed to examine the mutagenic specificity of (+)anti-dibenz[a,j]anthracene 3,4-diol-1,2-epoxide ((+)anti-DB[a,j]A-DE) in SOS-induced repair-proficient Escherichia coli ES87 (delta pro-lac, strA)/F' (pro+, lac1Q, lac1am26, lacZ delta M15). The plasmid pUB3, which contains the mutation target gene, supF, was modified with (+)anti-DB[a,j]A-DE in vitro (two to five adducts/plasmid) and then transformed into bacteria by electroporation. The spontaneous mutation frequency for unmodified pUB3 in uninduced cells was about 2 x 10(-6) and for SOS-induced cells, about 8 x 10(-6). The spontaneous supF- mutations were primarily insertions, deletions, and frameshifts. The mutation frequency for (+)anti-DB[a,j]A-DE-modified pUB3 was about 8 x 10(-6) and about 32 x 10(-6) for uninduced cells and SOS-induced cells, respectively. (+)anti-DB[a,j]A-DE induced primarily point mutations in supF in SOS-induced cells. GC-->AT transitions were the major mutations observed in SOS-induced cells (37%). GC-->TA (21%) and GC-->CG (8.6%) transversion mutations were also observed, whereas mutations at AT base pairs were rare (1.9%). Furthermore, a large number of tandem GC/GC-->AT/AT transition mutations were also observed (about 15% of all mutations in SOS-induced cells). Taken together, single and tandem GC-->AT mutations accounted for slightly over half (about 51%) of the mutations observed in SOS-induced cells. These results demonstrated that (+)anti-DB[a,j]A-DE was mutagenic in repair-proficient E. coli; however, unlike other polycyclic aromatic hydrocarbons that induce primarily transversion mutations, (+)anti-DB[a,j]A-DE caused mostly GC-->AT transitions.

Base Sequence↗
PubMed1993

Mutational specificity of the (+)-anti-diol epoxide of benzo[a]pyrene in a supF gene of an Escherichia coli plasmid: DNA sequence context influences hotspots, mutagenic specificity and the extent of SOS enhancement of mutagenesis.

Mutagenesis by the suspected major mutagenic metabolite of activated benzo[a]pyrene, which is (+)-anti-BPDE, was analyzed with a new system, which permits the selection of supF- mutations in an Escherichia coli plasmid using lactose minimal plates. (+)-anti-BPDE enhances base pairing mutations--principally at G:C base pairs, frameshift mutations and large deletions. Frameshift mutagenesis principally involves deletions and insertions of a single G:C base pair in runs of G:C base pairs. Base pairing mutations are significantly enhanced by SOS induction, especially GC-->TA mutations. Nearest neighbor analysis was performed assuming that a guanine (underlined) is being mutated, and (+)-anti-BPDE base pairing mutagenesis is enhanced by SOS induction in 5'-(A/T)G-3' sequences approximately 4-fold more than in 5'-(G/C)G-3' sequences, and in 5'-G(C/G)-3' sequences approximately 4-fold more than in 5'-G(A/T)-3' sequences; this is discussed. The influence of sequence context on quantitative aspects of (+)-anti-BPDE mutagenesis is considered, and hotspots are found at most, but not all, 5'-GG-3' sequences. The influence of sequence context on qualitative aspects of (+)-anti-BPDE mutagenesis (i.e. mutagenic specificity) is also considered. For example, the sequences, 5'-AG-3', 5'-CG-3' and 5'-GG-3', all have examples of G-->T, G-->A and G-->C mutations, while in the sequence, 5'-TG-3', only G-->T mutations have been detected. (The latter finding correlates with a recent site-specific study on the major adduct of (+)-anti-BPDE formed at N2-Gua in a 5'-TG-3' context, where G-->T mutations predominated [Carcinogenesis (1992) 13, 1415-1425].) These results suggest that sequence context plays a role in defining the kind of mutation (i.e. GC-->TA versus GC-->AT versus GC-->CG) induced by (+)-anti-BPDE, where the base on the 5' side of the guanine undergoing mutation seems to be influential. The most likely model for this is that sequence context influences adduct conformation, which controls mutagenic specificity.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1992

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: insertion elements dominate the spontaneous spectra.

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI- (am26), and carry the lacZ delta M15 marker for alpha-complementation in beta-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-alpha, the lactose operon is repressed (off). Furthermore, supF suppression of lacIam26 results in a lactose repressor that has an uninducible, lacIS genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF- mutations in pUB3 prevent suppression of lacIam26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is approximately 0.7 and approximately 1.0 x 10(-6) without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Elements; IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for -SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively). The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.

Base Sequence↗
PubMed1992

Mutagenesis by (+)-anti-B[a]P-N2-Gua, the major adduct of activated benzo[a]pyrene, when studied in an Escherichia coli plasmid using site-directed methods.

The suspected major mutagenic adduct of benzo[a]pyrene, (+)-anti-B[a]P-N2-Gua, is built into the unique PstI recognition site of the Escherichia coli plasmid, pUC19, in order to study its mutagenic potential. The adduct can either be at G437, which is replicated during leading strand DNA synthesis, or at G438, which is replicated during lagging strand DNA synthesis. The DNA strand complementary to the strand containing the (+)-anti-B[a]P-N2-Gua adduct is saturated with UV lesions to minimize its potential to generate progeny. Although all in-frame mutations could have been detected, a G437----T transversion mutation is virtually exclusively obtained at a frequency of approximately 0.04% per adduct following transformation into Uvr+ E. coli when SOS is not induced, and approximately 0.18% when SOS is induced. The mutation frequency of the adduct in a Uvr- background is estimated to be approximately 0.2% when SOS is not induced, and approximately 0.9% when SOS is induced. The absence of G438----T mutations is rationalized. G----T mutations from (+)-anti-B[a]P-N2-Gua are compared to the mutational specificity of the ultimate mutagenic form of activated benzo[a]pyrene.

Base Sequence↗
PubMed1991

Construction of a human shuttle vector containing a single nitrogen mustard interstrand, DNA-DNA cross-link at a unique plasmid location.

DNA cross-linking reagents are frequently unusually cytotoxic, and many, including the nitrogen mustards, are potent chemotherapeutic agents, presumably because DNA cross-links effectively block DNA replication. Most of these reagents form both inter- and intrastrand DNA cross-links, but it is unknown which is more effective at blocking replication and why. To evaluate the role of interstrand cross-links, a human shuttle vector was constructed that contains a single, nitrogen mustard interstrand cross-link at a unique site. In previous work (J.O. Ojwang, D. A. Grueneberg, and E. L. Loechler, Cancer Res., 49: 6529-6537, 1989) a duplex oligonucleotide was synthesized that had an interstrand cross-link derived from a nitrogen mustard moiety bound at the N(7)- position of the guanines in the opposing strands of a 5'-GAC-3' 3'-CTG-5' sequence. Herein, a procedure is described to incorporate this oligonucleotide into an SV40-based human shuttle vector, which was designed for these experiments. The purified cross-linked vector was characterized and shown: (a) to have a chemical (i.e., a nitrogen mustard) modification at the anticipated genome location; (b) to have a modification that covalently joins the two duplex strands of the vector together; and (c) to contain a single interstrand cross-link per genome. The methodologies described to construct this vector are expected to be generally applicable and, thus, site-specific incorporation of an interstrand cross-link derived from any appropriate chemical should be possible. These procedures complement existing methodologies that permit the incorporation of monoadducts and intrastrand cross-links into vectors in a site-specific manner.

Base Sequence↗
PubMed1991

Rotation about the C6-O6 bond in O6-methylguanine: the syn and anti conformers can be of similar energies in duplex DNA as estimated by molecular modeling techniques.

O6-Methylguanine (O6MeGua) is generally regarded as the most important premutagenic lesion formed from carcinogenic methylating agents, so its structure and mechanism of action have received considerable attention. Two conformations for the methyl group in O6MeGua are possible: in one the methyl group is syn with respect to the N(1)-position of the purine, points into the helix, and disrupts hydrogen bonding potential; in the second the methyl group is anti with respect to the N1-position of the purine, and points into the major groove. Syn-O6MeGua has been observed when paired with thymine in duplex DNA as determined by NMR, while anti-O6MeGua has been observed when paired with thymine in X-ray diffraction studies. Herein, molecular modeling/computational chemistry is used to evaluate this apparent discrepancy. [N6-Methyladenine (N6MeAde) was also studied, because it is isoelectronic with O6MeGua, and because more information is available about its energetics. Syn-N6MeAde is computed to be favored in small molecules; however, the fraction of the anti-conformer is computed to be approximately 7%, which agrees well with experimentally determined values (4-12%). In contrast, the anti conformation for N6MeAde is calculated to be favored in duplex DNA, which is consistent with what has been observed experimentally using both NMR and X-ray diffraction techniques. The agreement between the calculated and experimentally determined results with N6MeAde suggest that the methods are reasonable.] For O6MeGua, a syn/anti ratio of approximately 10(3) is computed for small molecules. In duplex DNA, syn-O6MeGua is computed to be favored, but the anti-conformer is less than approximately 1 kcal/mol higher in energy. Whether syn- or anti-O6MeGua predominates may depend upon sequence context, as well as environmental factors. The comparison between O6MeGua and N6MeAde suggests a rationale for the puzzling observation that O6MeGua appears to be a cytotoxic lesion in eukaryotic, but not prokaryotic, cells.

Base Sequence↗
PubMed1989

Synthesis of a duplex oligonucleotide containing a nitrogen mustard interstrand DNA-DNA cross-link.

Many cancer chemotherapeutic agents react with DNA and give adducts that block DNA replication, which is thought to result in cytotoxicity, especially in rapidly proliferating cells such as cancer cells. One class of these agents is bifunctionally reactive (e.g., the nitrogen mustards) and forms DNA-DNA cross-links. It is unknown whether inter- or intrastrand cross-links are more effective at blocking DNA replication. To evaluate this, a DNA shuttle vector is being constructed with an interstrand cross-link at a unique site. In the first step of this project, a duplex oligonucleotide containing an interstrand cross-link is isolated by denaturing polyacrylamide gel electrophoresis from the reaction of nitrogen mustard with two partially complementary oligodeoxynucleotides. The purified oligonucleotide product is characterized and shown to be cross-linked in a 5'-GAC-3' 3'-CTG-5' sequence by a nitrogen mustard moiety that is bound at the N(7)-position of the guanines in the opposing strands; the glycosylic bonds of these guanine adducts are stabilized in their corresponding imidazole ring-opened form. Nitrogen mustard is shown to react with a variety of oligonucleotides and, based upon these results, its preferred targets for interstrand cross-linking are 5'-GXC-3' sequences, where X can be any of the four deoxyribonucleotide bases.

Chemical Phenomena↗
PubMed1989

Adduct-induced base-shifts: a mechanism by which the adducts of bulky carcinogens might induce mutations.

Most carcinogens have been shown to be mutagens, and DNA adducts are formed when mutagenic/carcinogenic substances react with DNA. It is generally believed these adducts (or their derivatives) induce misreplication events that result in mutations. Many of the more potently mutagenic substances are bulky and three-dimensionally complex, such as the polycyclic aromatic hydrocarbons, aromatic amines, and aflatoxins; little is known about the mechanisms by which they induce mutations. Several theories exist and herein an additional mechanism is proposed by which bulky adducts might induce mutations at GC base pairs. Molecular modeling in conjunction with molecular mechanical calculation is used to assess if the mutagen/carcinogen moiety of the adduct might be able to shift the position of the base moiety of the adduct in such a way that misreplication events might be facilitated. This mechanism is referred to as adduct-induced base-shift, and two classes appeared possible; adduct-induced base-wobble and adduct-induced base-rotation. The latter has been proposed previously. By adduct-induced, base-wobble, the mutagen/carcinogen moiety of the adduct induces a shift in the position of the base moiety of the adduct with respect to the helix axis, which might facilitate mispairing events that are reminisent of non-Watson/Crick pairing that occurs at the wobble base of tRNA during translation. For example, in some guanine adducts, the guanine appears more thymine-like, which might facilitate G.A mispairing and thereby ultimately GC to TA transversion mutations. Adduct-induced base-rotation involves the rotation of the adducted base from the anti to the syn conformation and a variety of mispairing events might result.

Carcinogens↗
PubMed1989

Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies.

The mutational specificity and genetic requirements for mutagenesis by 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), one of the principal DNA lesions induced by oxidation and ionizing radiation, has been investigated in Escherichia coli. Thymine glycol was positioned at a unique site in the single-stranded genome of a bacteriophage M13mp19 derivative. Replication of the genome in E. coli yielded targeted mutations at a frequency of 0.3%; the mutations were exclusively T----C. Mutagenesis was independent of SOS and nth (nth encodes endonuclease III, a thymine glycol repair enzyme). The adduct was not detectably mutagenic in duplex DNA. A chemical rationalization for the mutation observed for thymine glycol was developed by applying molecular modeling and molecular mechanical calculations to the same DNA sequence studied in vivo. Modeling suggested that the 5R,6S isomer of cis-thymine glycol, when not base paired, was displaced laterally by approximately 0.5 A toward the major groove in comparison to the position that thymine would otherwise occupy. This perturbation of DNA structure should increase the likelihood of a guanine.thymine glycol wobble base pair during replication, which would explain the mutational specificity of the base observed in the genetic experiments.

Base Sequence↗
PubMed1989

Mutagenic specificity of alkylated and oxidized DNA bases as determined by site-specific mutagenesis.

This work demonstrates the use of the tools of site-specific mutagenesis to study the mutagenic activity of two DNA adducts, O6-methylguanine and cis-thymine glycol. The former adduct is one of the methylated bases formed by carcinogenic and mutagenic alkylating agents. It was built into the single-stranded genome of bacteriophage M13 and replicated in Escherichia coli (E. coli). The mutation frequency of O6-methylguanine was 0.4% in physiologically normal cells. In cells in which the repair systems for O6-methylguanine were compromised by challenge with an alkylating agent, the mutation frequency rose to approximately 20%. DNA sequencing revealed that O6-methylguanine induced exclusively G----A transitions, which was most consistent with it pairing with thymine during DNA synthesis. The mutagenic effects also were investigated of cis-thymine glycol isomers, which are major, stable products of ionizing radiation and oxidative damage to DNA. By techniques similar to those employed for the study of O6-methylguanine mutagenesis, a single thymine glycol was situated in an M13 phage genome. The genome was replicated in E. coli that were physiologically normal, induced for SOS functions, or deficient in the nth gene product and, in all cases, the mutagenic processing of thymine glycol in vivo yielded mutant progeny phage at a frequency of 0.3-0.4%. All mutations occurred at the site that originally contained thymine glycol, and all were demonstrated by DNA sequencing to have resulted from targeted T----C transitions. These data suggest that thymine glycol pairs with guanine during replication.

DNA Mutational Analysis↗
PubMed1988

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

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

Aflatoxin B1↗
PubMed1988

Mapping the binding site of aflatoxin B1 in DNA: molecular modeling of the binding sites for the N(7)-guanine adduct of aflatoxin B1 in different DNA sequences.

Aflatoxin B1 (AFB1), a potent mutagen and carcinogen, forms an adduct exclusively at the N(7) position of guanine, but the structure of this adduct in double stranded DNA is not known. Molecular modeling (using the program, PSFRODO) in conjunction with molecular mechanical calculation (using the program, AMBER) are used to assess the binding modes available to this AFB1 adduct. Two modes appear reasonable; in one the AFB1 moiety is intercalated between the base pair containing the adducted guanine and the adjacent base pair on the 5'-side in reference to the adducted guanine, while in the second it is bound externally in the major groove of DNA. Rotational flexibility appears feasible in the latter providing four, potential binding sites. Molecular modeling reveals that the binding sites around the reactive guanine in different sequences are not uniformly compatible for interaction with AFB1. As the sequence is changed, one particular external binding site would be expected to give a pattern of reactivities that is reasonably consistent with the observed sequence specificity of binding that AFB1 shows in its reaction with DNA (Benasutti, M., Ejadi, S., Whitlow, M. D. and Loechler, E. L. (1988) Biochemistry 27, 472-481). The AFB1 moiety is face-stacked in the major groove with its long axis approximately perpendicular to the helix axis. Favorable interactions are formed between exocyclic amino groups that project into the major groove on cytosines and adenines surrounding the reactive guanine, and oxygens in AFB1; unfavorable interactions involve van der Waals contacts between the methyl group on thymine and the AFB1 moiety. "Some of the sequence specificity of binding data can be rationalized more readily if it is assumed that 5'-GG-3' sequences adopt an A-DNA structure." Based upon molecular modeling/potential energy minimization calculation, it is difficult to predict how reactivity would change in different DNA sequences in the case of the intercalative binding mode; however, several arguments suggest that intercalation might not be favored. From these considerations a model of the structure for the transition state in reaction of AFB1 with DNA is proposed involving one particular external binding site.

Aflatoxin B1↗