Insights into the Conformations of Arylamine

Insights into the Conformations of Arylamine-DNA Adduct
in Various Replicative & Repair Systems
Arylamines are a well-known group of mutagens/carcinogens, which have been implicated in the
etiology of several sporadic human cancers. We have investigated arylamine-induced DNAadduct conformational heterogeneity using various gel, spectroscopic (NMR, CD) and chip(SPR) and microcalorimetry-based (DSC, ITC) methods. The active site conformation of the
mutagenic amine-dG adduct was studied in the presence of Klenow fragment of E. coli DNA
polymerase I (Kfexo–) and DNA polymerase  (pol ) using 19F NMR, insertion assay, and
surface plasmon resonance (SPR). Kfexo– prefers an open downstream single-stranded template,
whereas pol  prefers single-nucleotide gapped substrates. In a single nucleotide gap, the
aminofluoriene-dG adduct adopts both B-type and stacked (S) conformation and this
heterogeneity is retained upon binding pol . Addition of a non-hydrolysable dCTP and dATP
analog to the binary complex results in an increase of B-type and wedge conformation,
respectively. In contrast, the same adduct at the replication fork of the Kfexo–/DNA complex
adopts a mixture of B and S with minimal effect upon addition of non-hydrolysable nucleotides.
For pol , the insertion of dCTP was preferred opposite the lesion in a single nucleotide gap
assay consistent with 19F-NMR data. SPR binding kinetics revealed that pol  binds tightly with
DNA in the presence of correct dCTP but the adduct weakens binding with no nucleotide
specificity. Similarly, we have probed the role of conformational heterogeneity for nucleotide
excision repair (NER) of the bulky arylamine adducts in E. coli. Our study indicates that lesioninduced DNA bending/thermodynamic destabilization is more important than the usual S/B
conformational heterogeneity, demonstrating the complexity in DNA recognition factors for
repair of bulky lesions. Taken together, these results provide conformational insights into the
DNA binding characteristics of a bulky arylamine lesion in the active site of DNA polymerases
and repair proteins.
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