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To quantitatively evaluate the extent of the blockage posed by N-dA PCL12 for the pol ν-catalyzed nucleotide insertion, dTTP titration assays (6.4 nM to 100 μM dTTP at 5-fold incrementally increased concentrations, left to right) were performed.
The primer extensions and single nucleotide incorporations showed some limited bypass of N-dA PCL12 at very high dNTP concentrations (100 μM), while the titration experiment with varying concentrations of dTTP (from 6.4 nM to 100 μM) revealed the severity of the blockage posed by this lesion for the pol δ-catalyzed nucleotide insertion.
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