4 research outputs found

    Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

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    The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo−) and DNA polymerase β (pol β) using 19F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2′-deoxycytosine-5′-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo− complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with 19F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential

    Biochemical Characterization of DNA Glycosylases from Mycobacterium Tuberculosis

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    The DNA glycosylases function in the first step of the base excision repair (BER) process, that is responsible for removing base lesions resulting from oxidation, alkylation or deamination. The DNA glycosylases that recognize oxidative base damage fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis. While Fpg proteins are widely distributed among the bacteria and plants, Nei homologs are sparsely distributed across phyla, and are only found in γ-proteobacteria, actinobacteria and metazoans. Interestingly, M. tuberculosis H37Rv harbors two proteins (Rv2464c and Rv3297) from the Nei clade and two (Rv2924c and Rv0944) from the Fpg clade. All four Fpg/Nei proteins were successfully overexpressed by using a novel bicistronic vector, which theoretically prevented stable mRNA secondary structure(s) surrounding the translation initiation region (TIR) thereby improving translation efficiency. Additionally, MtuNth (Rv3674c) was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligonucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from γ-irradiated DNA. MtuFpg1 (Rv2924c) has a substrate specificity similar to that of EcoFpg and recognizes oxidized purines. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG); however, MtuFpg1 has a substantially increased opposite base discrimination compared to EcoFpg. The Rv0944 gene encodes MtuFpg2, which contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 (Rv2464c) recognizes oxidized pyrimidines not only on doublestranded DNA but also on single-stranded DNA. It also exhibits uracil DNA glycosylase activity as well as weak activity on FapyA and FapyG. MtuNth recognizes a variety of oxidized bases, such as urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5- OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd) as well as FapyA, FapyG and 8-oxoadenine (8-oxoA). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers of Tg, whereas MtuNth recognizes only the (5S) isomers. The other Nei paralog, MtuNei2 (Rv3297), did not demonstrate activity in vitro as a recombinant protein, but when expressed in Escherichia coli, the protein decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs. Since pathogenic bacteria are often exposed to an oxidative environment, such as in macrophages, our data, together with previous observations, support the idea that the BER pathway is of importance in protecting M. tuberculosis against oxidative stress, as has been observed with other pathogens

    Mechanistic investigations of DNA reactive carcinogens at low dose, through analysis of DNA adducts, mutations and DNA repair.

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    Genetic toxicology assesses the genotoxic potential of chemicals in consumer products, pharmaceuticals and from agricultural and industrial processes. Such assessment is integral in hazard identification and risk assessment to prevent unnecessary human exposure and limit cancer risk. Human risk assessments for genotoxic alkylating agents were based upon linear dose-response models where genotoxicity accrues proportionally with dose. Evidence is accumulating to support a non-linear dose-response at low doses of ethyl methanesulfonate (EMS), a model alkylating agent. For acceptance of non-linear dose responses, a strong explanatory mechanism of action needs to be elucidated. In the following work, low dose mutagenic effects of methyl nitorosurea (MNU), the most potent alkylating agent, have been examined in AHH-1 human lymphoblastoid cells using the HPRT assay. An increase in mutant frequency was not observed until 0.01pg/ml MNU (LOGEL, Lowest Observed Genotoxic Effect Level) with a No-Observed Genotoxic Effect Level (NOGEL) at 0.0075pg/ml MNU. Of interest, is the apparent hormesis induced at 0.0025pg/ml MNU. The principle adduct responsible for MNU mutagenesis is 0 6Methylguanine (06MeG) that miscodes during replication and becomes fixed as GC->AT transitions. Accordingly, the non-linear increase in mutant frequency is accompanied by a non-linear increase in GC->AT transitions. Furthermore, evidence is provided that implicates methlyguanine methyltransferase (MGMT) in protecting DNA from MNU induced mutagenesis by repairing 0 6MeG at low doses, thereby creating the NOGEL. AHH-1 cells treated with 0 6Benzylguanine (06BG), to inactivate MGMT, were hypersensitive to low dose MNU mutagenesis. At 0.0075pg/ml MNU, there was a three-fold increase in mutant frequency and an increase in proportion of GC-^AT transitions, from 28% to 48% in MGMT inactivated cells. This thesis presents a non-linear dose-response for MNU with a strong biological mechanism of action involving DNA repair
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