Backbone Conformational Equilibrium in Mismatched DNA Correlates with Enzyme Activity

Abstract

T:G mismatches in mammals arise primarily from the deamination of methylated CpG sites or the incorporation of improper nucleotides. The process by which repair enzymes such as thymine DNA glycosylase (TDG) identify a canonical DNA base in the incorrect pairing context remains a mystery. However, the abundant contacts of the repair enzymes with the DNA backbone suggest a role for protein-phosphate interaction in the recognition and repair processes, where conformational properties may facilitate the proper interactions. We have previously used ³¹P NMR to investigate the energetics of DNA backbone BI-BII interconversion and the effect of a mismatch or lesion compared to canonical DNA and found stepwise differences in ΔG of 1-2 kcal/mol greater than equivalent steps in unmodified DNA. We have currently compared our results to substrate dependence for TDG, MBD4, M. HhaI, and CEBPβ, testing for correlations to sequence and base-pair dependence. We found strong correlations of our DNA phosphate backbone equilibrium (K_eq) to different enzyme kinetics or binding parameters of these varied enzymes, suggesting that the backbone equilibrium may play an important role in mismatch recognition and/or conformational rearrangement and energetics during nucleotide flipping or other aspects of enzyme interrogation of the DNA substrate.

Figure 1

(A) Definition of the backbone dihedral angles most associated with the BI–BII equilibrium (ε and ζ). (B) Illustration of the putative BI–BII interconversion equilibrium energy diagram, the backbone conformations, and the definition of the Gibbs free energies associated with the equilibrium with respect to the forward process.

Figure 2

Stacked 1D ³¹P spectra for our five non-palindromic DNA duplexes at 10 °C. Starting at the bottom is our control sequence with a CpG:C context (blue), followed by ApG:T (purple), GpG:T (cyan), TpG:T (pink), and CpG:T (yellow). They are ordered with increasing TDG activity, and their values for k_max are given. These spectra have been evaluated for all phosphates, with the three flanking the mismatched T indicated by colored arrows: G3pC/T4 (blue), C/T4pX5 (orange), and X5pT6 (black). Note that the T4pX5 phosphate (orange arrows) in the T:G mismatches has dramatic sequence dependence, whereas the others do not. See Table 1 for complete duplex identity. C/T refers to sequence positions that differ by having a C in a control DNA and a T as a mismatch at the same position.

Figure 3

Comparison of ΔG (A) and ΔΔG (B, defined as ΔG_ctrl – ΔG_test) as a function of phosphate position (horizontal axis), temperature (bars) from 5 to 20 °C (except TpG:T, where the 20 °C experiment gave poor data), and sequence dependence. The mismatched T and base-paired G are indicated by arrows. The boxes highlight the phosphate positions flanking the mismatched T, demonstrating the significant sequence dependence. Note that the 3′ ends are eliminated due to melting issues at the higher temperatures. Uncertainty in ΔG values was determined to be ±0.01 kcal/mol based upon standard error propagation from 0.02 ppm uncertainty in ³¹P isotropic chemical shift values.

Figure 4

Minimal kinetic mechanism as adapted from Dow et al. Copyright 2019 American Chemical Society.

Figure 5

Correlations between DNA backbone equilibrium (ln K_eq or K_eq) for the T4pX5 phosphate and TDG activity as a function of sequence contexts. (A) WT TDG, (B) Q278A TDG mutant, (C) A145G TDG mutant, (D) A145G TDG mutant comparing K_eq to K_flip. All data points have error bars representing the standard deviation of both the NMR data plus the data provided in the original manuscripts. All TDG data adapted from ref (24). Copyright 2019 American Chemical Society. Note the different axes arise due to the different systems and properties being analyzed.

Figure 6

Correlations between DNA backbone equilibrium (K_eq or ln K_eq) and enzyme activities as a function of base-pairing context. (A) WT TDG k_max. Adapted with permission from refs (22) and (23). Copyright 2006 American Chemical Society and CC BY 4.0, respectively. (B) MBD4 k_obs. Adapted with permission from ref (25). Copyright 2012 Elsevier (C) M. HhaI relative affinity R. Adapted with permission from ref (43). Copyright 1995 Oxford University Press. (D) CEBPβ binding constant (K_D). Adapted with permission from ref (30). Copyright 2021 Oxford University Press. All data points have error bars representing the standard deviation of both the NMR data plus the data provided in the original manuscripts. Note that M:G refers to a 5-methyl-cytosine:guanine base pair. Note the different axes arise due to the different systems and properties being analyzed.