Date of Award
Doctor of Philosophy in Pharmaceutical Sciences
Biomedical and Pharmaceutical Sciences
Cancer is a collection of diseases defined by uncontrolled cell growth and dissemination of abnormal cells. It is the second most deadly disease in the United States. Exposure to chemicals, both endogeneous and exogeneous is one of the major causes of cancer initiation. The role of chemicals in carcinogenesis was observed back in the eighteenth century when surgeon Pott observed a large number of scrotal cancer cases in chimney sweeps and associated it with to the soot exposure. However, research in the field of chemical carcinogenesis was renewed when Yamagawa and Ichikawa reported the development of malignant tumor on the rabbit’s ear due to the regular application of coal tar. Later, it was discovered that the presence of the polycyclic aromatic hydrocarbon benzo[a]pyrene in the coal tar was responsible for its tumorogenicity. Following that finding, researchers have made significant advancement in the field of chemical carcinogenesis.
Majority of the chemical carcinogens are genotoxic in nature which exert their effect primarily by reacting with the DNA. These chemicals are either reactive as such or undergo enzymatic activation into the electrophilic species that enable them to form covalent adducts with the electron-rich DNA. Therefore, DNA-adduct formation is considered as a hallmark of chemical carcinogenesis. DNA-adducts are formed regularly in the cells as the genomic DNA is under regular attack by various chemicals. Cells have developed various repair mechanisms such as nucleotide excision repair (NER), base excision repair (BER) to protect the DNA from the effect of these adducts. Despite the effectiveness of these defense mechanisms some of the adducts have the potential to evade them. Unrepaired adducts can enter in the replication cycle and affect the process which creates a possibility of various kinds of mutation induction. Mutation on the genes that control the cell growth may trigger cancer initiation. For example, mutation of tumor suppressor p53 gene is regularly seen in the majority of sporadic human cancers.
Arylamines is an important group of chemical carcinogens that are implicated in the etiology of human cancers. 2-Naphthylamine, benzidine, 4-aminobiphenyl and 2-acetylaminofluorene are among the well-known arylamine carcinogens. 2-Acetylaminofluorene was originally developed as a pesticide, but was banned because of its liver carcinogenicity on animals. However, it is used continuously by researchers as a model carcinogen to study the chemical carcinogenesis. Arylamines are not reactive per se but undergo metabolic transformations in the body to produce highly reactive nitrenium ion, which bind with DNA specifically at C-8 position of guanine to yield two major C8-substituted dG adducts: N-(2'-deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF). Similarly, bladder carcinogen 4-aminobiphenyl forms N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). The NMR studies on ABP, AF, and AAF adducted duplexes found that they exist in a mixture of two prototype conformers; anti-glycosidic B-conformer in which the fluorenyl moiety orients in the major groove of double helical DNA without affecting the Watson-Crick base pairing and syn-glycosidic stacked-conformer (S) in which the carcinogen is inserted into the duplex resulting into flipping out of the modified guanine. Along with the above two conformers, AAF additionally produces a syn-glycosidic wedge-conformer (W) with the fluorene moiety in the narrow minor groove of the duplex. The population of these conformers was found to be dependent on the nature of the lesion and the bases surrounding it.
Arylamine-DNA adducts are good substrates for NER, a repair pathway responsible for removing bulky lesions. The repair efficiency of NER proteins are found to be affected by different chemical, structural and conformational factors. The nature of bases surrounding the lesion site is an important factor which is generally stated as sequence effect. The NER of arylamines exhibits dramatic sequence effects. One of the most significant effect was witnessed on the reparability of AAF adduct involving E. coli UvrABC and human exonuclease NER systems, where the lesion presents separately on each of the three guanines of the NarI sequence (5'-…CG1G2CG3CC...-3'). It was discovered that in E. coli and human exonuclease the relative repair efficiencies of AAF at G1, G2 and G3 were in a ratio of 100:18:66 and 38:100:68, respectively. But, the rationale behind this sequence dependent modulation of NER is not clear.
We hypothesize that adduct induced conformational heterogeneity is modulated by the base sequences around the lesion and the thermal/thermodynamic stability of the lesion containing DNA duplexes is an important factor for determining their repair efficiencies. In Manuscript I (published in Nucleic Acids Research), the objective was to test our hypothesis on the aforementioned sequence dependent NER of AAF in the NarI hot spot sequence. To that end, we prepared three 16-mer oligonucleotide duplexes that are site specifically modified by FAAF (fluorinated derivative of AAF) at G1, G2 or G3 of the NarI sequence (5ꞌ-CTCTCG1G2CG3CCATCAC-3ꞌ). We utilized 19F NMR and circular dichroism (CD) to determine the conformational profiles of the FAAF in the three duplexes. We conducted melting experiments using UV-spectrophotometer and differential scanning calorimetry (DSC) for thermodynamic analyses. In addition, we carried out NER assay of these duplexes using the E. coli UvrABC system. Similarly, the repair work was also performed on FAF (fluorinated derivative of the N-deacetylated AF) modified duplexes as their conformational and thermodynamic properties were known.
Our 19F NMR/CD data showed that the FAAF at G1 and G3 orients majorly in syn-glycosidic S- and W-conformers, whereas the anti glycosidic B-conformer is favored at G2. The thermodynamic data obtained from UV and DSC indicates that the S- and W-conformers induce greater distortion and thermodynamic destabilization of the modified duplexes. Interestingly, we found that the repair of FAAF occurred in a conformation-specific manner with highly S/W-conformeric AAF at G3 and G1 incised more efficiently than the B-type G2 (G3~G1 > G2). The N-deacetylated AF adducts in the same NarI sequence were repaired 2-3 fold less than the bulky N-acetylated FAAF, but the order of reparability was different (G2~G1 > G3), a reverse order of the FAAF. We believe the presence of N-acetyl group raised the conformational barrier of FAAF vs. FAF. Overall, the results provided conformational and thermodynamic insight into the sequence dependent UvrABC incisions of the bulky aminofluorene DNA adducts.
In Manuscript II (to be submitted in Chemical Research in Toxicology), we extended our NER mono-adducts work (Manuscript I) to FAAF derived di-adducts formed from the NarI sequence. The objective was to understand how the cluster adducts are treated by the NER proteins and to investigate the sequence effects in the repair efficiency of the arylamine di-adducts, if any. To do so, we prepared modified oligonucleotides that contain FAAF simultaneously on two (G1G2, G2G3 and G2G3) of the three guanines (G1, G2, and G3) of the NarI sequence (5ꞌ-CTCTCG1G2 CG3CCATCAC-3ꞌ). We conducted the NER assays on the three di-adducts using the E. coli UvrABC proteins. Moreover, we performed spectroscopic (19F NMR and CD) and melting experiments for conformational and thermodynamic examination, respectively.
We found that the repair of the bulky FAAF di-adducts occurred with double the efficiency of the mono-adducts. Our structural data related the enhanced reparability with the greater thermal and thermodynamic destabilization produced in the di-adduct duplexes. In addition, the three di-adduct duplexes exhibited dramatic sequence effects in the repair efficiency. Although, the 19F NMR data was not conclusive to provide their conformational profile, the respective mono-adducts data (Manuscript I) indicate that the repair seems to occur in a conformation-specific manner. The NarI-G2G3 and -G1G3 di-adducts are repaired more efficiently and they contain FAAF at G3 position, in which FAAF is known to exist in highly S/W-conformation. In contrast, the NarI-G1G2 di-adduct contains the highly B-conformeric G2 position. Apparently, duplex distortion by destacking and destabilization is important as the repair and destacking/destabilization trend followed in the same order: G2G3 > G1G3 > G1G2. The results of this study provide the first report on the structure-repair relationships of FAAF cluster adducts.
Arylamine adducts are also capable of inducing mutation. For example, AF produces point mutation and AAF results into a mixture of point and frameshift mutations. Aforementioned sequence effects in the NarI sequence are not limited to repair, but were also seen in the mutational outcome. Previously, it has been reported that AF and AAF adducts yield significantly higher frequency of -2 frameshift mutation only when adducted to third guanine (G3) of the NarI sequence (5ꞌ--G1G2CG3CN--3ꞌ). In addition, the mutational rate was found to be governed by the base at N position. The presence of cytosine (C) at position N resulted into relatively higher rate of mutation as compared to thymine (T). Structural studies from our laboratory have shown that the FAF adduct adopts stacked (S) conformer when N is = C, but displayed conformational heterogeneity when C is replaced by T. In Manuscript III (published in Nucleic Acids Research), our focus was to determine whether the above 3ꞌ-next flanking base (N) effect on the conformational property is limited to AF modified NarI sequence or can also exist in random G*CN duplexes modified by different arylamines (ABP, AF, AAF).
We prepared two 11mer duplexes (5ꞌ-CCATCGCNACC-3ꞌ, N= T or A) having the same G*CN sequence context as in NarI sequence and modified them with FABP, FAF or FAAF. We utilized various techniques including 19F NMR, CD, DSC, gel electrophoresis (EMSA) to study the structural properties of the two duplexes. In addition we performed molecular dynamics/potential of mean force (MD/PMF) calculations. We also performed E.coli UvrABC based NER assays to determine the NER significance of 3ꞌ-next flanking base (N) effect. Our results showed that the A to T polarity swap in the G*CA/G*CT transition produced a dramatic increase in destabilized stacked conformation, but resulted in unexpected 2~3-fold lower NER efficiencies. However, the results were consistent with lesion-induced DNA bending/distortion determined by CD, EMSA and MD/PMF calculations. As for the lesions, FAAF displayed 3~4 times better reparability than FABP and FAF lesions, which is consistent with the magnitude of bending and helix destabilization. Overall, the results suggest that lesion-induced DNA bending/thermodynamic destabilization is a more important NER factor than the usual S/B conformational heterogeneity, as has been observed previously for AF and AAF in certain sequence contexts.
In Manuscript IV (to be submitted in Chemical Research in Toxicology), we extended our previous work (Manuscript III) to examine the effect of 3ꞌ-next flanking T induced conformational heterogeneity on the DNA polymerase mediated translesion synthesis (TLS). Initially, we carried out chemically simulated TLS (without DNA polymerase) to deduce the conformational property (using 19F NMR and CD) of FAF/FABP adducts at the replication fork and during primer elongation across modified G*CA and G*CT templates. Following conformational analysis, we performed DNA polymerase mediated running start and steady state kinetics experiments to probe the effect on the insertion efficiency and fidelity of DNA polymerases. FAF and FABP in the G*CA and G*CT sequences were found to adopt a similar conformational profiles at the replication fork which is in contrast to the usual duplex. Due to the similar conformational profiles, no substantial 3ꞌ-next flanking base effect was observed in the base insertion efficiency at the replication fork. However, we observed some lesion effect in polymerase stalling around the lesion site, i.e., the anti-conformer FABP showed more stalling at the preinsertion site (n-1) whereas the syn-conformeric FAF at the lesion site (n). Overall, there was no significant 3ꞌ-next flanking base effect on the activity of polymerases. However, the results provide valuable insights on the role of lesion-induced conformational heterogeneity in modulating the translesion DNA synthesis.
In summary, the studies described in this Thesis provided the structural and thermodynamic rationales for the sequence- and lesion-dependent repair and replication of the bulky arylamine-DNA adducts. In addition, we demonstrated how the global duplex distortion overpowers the local conformational disturbances as an important damage recognition factor. To the best of our knowledge, this is the first report on the structure and repair characteristics of arylamine derived cluster adducts along with the dramatic sequence effects. Our results also provided a glimpse of replication across the arylamine adduct and the role of adduct conformation in modulating the process of TLS. Overall, we established a strong and sensible structural-function relationships between the structure/conformation of arylamine adducts and their mutational and repair outcomes.
Jain, Vipin, "Sequence Effects on the Repair and Replication of Arylamine-DNA Adducts" (2013). Open Access Dissertations. Paper 22.