LAUSR

T:G mismatches in DNA result in humans primarily from deamination of methylated CpG sites. They are repaired by redundant systems such as thymine DNA glycosylase (TDG) and methyl-binding domain enzyme (MBD4), and maintenance of these sites has been implicated in epigenetic processes. The process by which these enzymes identify a canonical DNA base in the incorrect base pairing context remains a mystery. However, the conserved contacts of the repair enzymes with the DNA backbone suggests a role for protein-phosphate interaction in the recognition and repair processes. We have used 31P NMR to investigate the energetics of DNA backbone BI-BII interconversion, and for this work have focused on alterations to the activation barriers to interconversion and the effect of a mismatch compared to canonical DNA. We have found alterations to the ΔG of interconversion for T:G base pairs are remarkably similar to U:G base pairs in the form of step-wise differences in ΔG of 1-2 kcal/mol greater than equivalent steps in unmodified DNA, suggesting a universality of this result for TDG substrates. Likewise, we see perturbations to the free energy (∼1 kcal/mol) and enthalpy (2-5 kcal/mol) of activation for the BI-BII interconversion localized to the phosphates flanking the mismatch. Overall our results strongly suggest that the perturbed backbone energetics in T:G base pairs play a significant role in the recognition process of DNA repair enzymes.