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

Abstract:

The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct [dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene] have been investigated in the presence of Klenow fragment of E. coli DNA polymerase I (Kfexo-) and DNA polymerase beta (pol beta) using 19F NMR, insertion assay, and surface plasmon resonance (SPR). 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 beta. Addition of a non-hydrolysable 2'-deoxy-cytosine-5-[(a,b)-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 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 addition of non-hydrolysable nucleotides. For pol beta, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with 19F-NMR data. SPR binding kinetics revealed that pol beta 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.

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