Date of Award
Doctor of Philosophy in Pharmaceutical Sciences
Biomedical and Pharmaceutical Sciences
Cancer is a major public health problem worldwide. An estimated 1.6 million new cases of cancer will be diagnosed in the United States in 2017. Most cancers are assumed to develop from the initiation of chemically induced DNA damages. Chemical carcinogens are genotoxic and they undergo enzymatic activation into reactive electrophilic species to form covalent bonds with the electron-rich DNA that form adducts. Aromatic amines are one of the most examined chemical carcinogens, and once activated, these amines attack the C-8 position of guanine to form C8-dG adducts (ABP [N-(2′-deoxyguanosin-8-yl)-2-aminobiphenyl], AF [N-(2′-deoxyguanosin-8-yl)-2-aminofluorene], and AAF [N-(2′-deoxyguanosin-8-yl)-2 acetylaminofluorene]); these formations result in DNA lesions. These bulky DNA lesions at the replication fork can induce a conformational heterogeneity, which complicates mutational outcomes. These outcomes are due to a single DNA adduct that can adopt multiple conformations at the replication fork, and a polymerase may process each conformation in different ways. Studies have shown differences in the mutational consequences of arylamine adducts in different DNA sequences. AF, AAF, and ABP all adopt anti B-DNA , syn stacked (S), and wedge (W) DNA conformational heterogeneity depending on sequence contexts. A replication polymerase is likely to stall or completely stop DNA synthesis when it encounters a bulky DNA lesion; this outcome allows recruitment of various bypass polymerases. This move will determine whether the synthesis is to be error free or error prone. Understanding the processes involving replication polymerases in arylamine-induced translesion synthesis is important; these processes are implicated in mutation and repair.
Our working hypothesis is that the sequence-dependent arylamine-conformational heterogeneity is an important consideration for its mutational and repair outcomes. In Manuscript I (to be submitted to Journal of Chemical Research in Toxicology), we explored the arylamine-conformational heterogeneity. We previously reported a 3′-next flanking sequence effect [G*CT vs. G*CA; G*, FABP, N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl; FAF, N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene] on arylamine-DNA lesions in relation to DNA replication and repair. In the present study, we present an unusual sequence effect on a 16-mer fully paired DNA duplex 5′-CTTCTG1G2TCCTCATTC-3′, in which the same lesion modification at G1 and G2 revealed a dramatic difference in S (base-displaced stacked)/B (major groove binding B-type) conformational heterogeneity. We obtained conformational (19F-NMR/CD), calorimetric (DSC and ITC), and binding (SPR) data for a set of four 16-mer modified templates (FABP vs. FAF and TG1*G2T vs. TG1G2*T) during primer elongation. Dynamic 19F-NMR results indicate that FABP modification at G1 and G2 resulted in 67%:33% of B:S and 100% B population ratios, respectively, and the planar FAF in the same sequence contexts exhibited 25%:75% B:S and 83%:17% B:S, respectively. A significant increase was observed in S-conformation (0% to 17%) going from a twisted nonplanar FABP to a planar FAF. Calorimetric (DSC and ITC) and UV-melting data revealed that the B-conformer is a thermodynamic stabilizer in duplex settings, and the S-conformer is a destabilizer. The situation is reversed during the primer elongation process that involves ss/ds duplexes, i.e., S-conformation promotes lesion stacking at the ss/ds replication fork. This finding is in agreement with SPR binding results, which showed that lesion stacking increases the binding affinity of the complementary strands in the order of FABP > FAF and G*GT > GG*T. Surprisingly, our data revealed that the twisted biphenyl is a better stacker at ss/ds junction than the coplanar FAF, possibly due to its ring flexibility that helps accommodate the replication fork structures. This finding is not the case for fully paired duplexes in which base-displaced stacking is enhanced by lesion planarity. The extent of primer dissociation rates across the lesion was influenced by the DNA sequences and the nature of the lesion (FAF vs. FABP). These results provide conformational insights into the important role of ss/ds sequence effects in modulating bulky lesion-induced translesion DNA synthesis. Manuscript II (to be submitted to Journal of Nature Structural & Molecular Biology) is the extension of manuscript I. Bulky organic carcinogens are usually activated in vivo and form DNA adducts. Some of these DNA adducts exist in multiple conformations that are slowly interconverted to one another. For example, the dG-C8-lesion of the human bladder carcinogen 4-aminobiphenyl (ABP) has been reported to exist in a sequence-dependent mixture of B/S conformations. Different conformations have been shown to lead to different kinetic capacities, binding capacities, and ultimately different translesion synthesis and transcription capacities. Although rare, conformation-dependent mutagenic and repair patterns have been reported. For example, the bulky Nacetylaminofluorene at G3 in the NarI sequence (5′-G1G2CG3CN-3′) induces a high frequency of −2 frameshift mutations, whereas the adducts on G1 and G2 are not mutagenic. Results show why mutagenic hotspots exist in the genome. However, the conformation-specific replication block, which is more relevant to replication, has not been reported. Such research deficiency may be due to the dynamicity of the replication block which usually detect either the ensemble of consequence of all the conformers or the culmination of all cellular behaviors, such as mutagenicity or survival rate. In Manuscript I, we showed very unusual sequence-dependent conformational heterogeneities involving FABP-modified DNA under different sequence contexts (TG1*G2T [67%B:33%S] and TG1G2*T [100%B], G*, FABP, N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl). Different biological consequences are believed to be linked to different adducts. In the present study, we attempted to correlate the replication block to different conformations from the single FABP-modified DNA lesion. We utilized a combination of biophysical (SPR and steady-state kinetics) tools to reveal the differences in vitro. The results were discussed in terms of conformational differences. Manuscript III (has been accepted by Journal of ACS Omega) is a technical paper. We focused on the oligonucleotides. Oligonucleotides serve as important tools for biological, chemical, and medical research. The preparation of oligonucleotides through automated solid-phase synthesis is well established. However, identification of byproducts generated from DNA synthesis, especially from oligonucleotides containing site-specific modifications, is sometimes challenging. Typical HPLC, Mass Spectrometry (MS), and gel electrophoresis methods alone are not sufficient for characterizing unexpected byproducts, especially for those having identical or very similar molecular weight (MW) to the products. We used a rigorous quality control procedure to characterize byproducts generated during oligonucleotide syntheses: 1) purify oligonucleotide by different HPLC systems; 2) determine the exact MW by high resolution MS; 3) locate modification position by MS/MS or exonuclease digestion with MALDI-TOF analysis; and 4) conduct, where applicable, enzymatic assays. We applied these steps to characterize byproducts in the syntheses of oligonucleotides containing important methyl DNA adducts 1-methyladenine (m1A) and 3-methylcytosine (m3C). In m1A synthesis, we differentiated a regioisomeric byproduct 6-methyladenine, which possesses identical MW to m1A. As for m3C, we identified a deamination byproduct 3-methyluracil, which is only 1 Dalton greater than m3C in the ~ 4,900 Dalton context. The detection of these byproducts would be very challenging if the abovementioned procedure were not adopted.
Cai, Ang, "Conformational Insights Into Aminobiphenyl-Modified DNA: Implications for Mutation and Repair" (2017). Open Access Dissertations. Paper 657.