Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site
Year: 2013
Type: article
Abstract: 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 19 F 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 19 F 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. Background: DNA lesions in different sequence contexts exhibit alternate conformations. Results: DNA adduct conformation is differentially modulated by DNA structure, DNA polymerase, and the presence of an incoming nucleoside triphosphate. Conclusion: Adduct conformation is influenced by binding of a non-hydrolysable nucleotide to pol β but not Klenow fragment. Significance: Adduct conformation and coding potential is modulated by constraints imposed by the polymerase active site.
Source: Journal of Biological Chemistry
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
Awards: Structure-activity Relationships of Hiv-1 Reverse Transcriptase, Mechanisms of Nucleic Acid Enzymes - Pol Beta and Mammalian Base Repair Enzymes, RI Network for Excellence in Biomedical and Behavioral Research, Sequence Effects of Arylamine-DNA Adducts: Repair and Replication, DNA Polymerase Fidelity Mechanisms: Theory and Experiment, G7025949813, Sequence Effects of Arylamine-DNA Adducts: Repair and Replication, DNA Polymerase Fidelity Mechanisms: Theory and Experiment
OpenAlex ID: W2087922772