Backbone Conformational Equilibrium in Mismatched DNA Correlates with Enzyme Activity

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 (Keq) 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 S1 .
Figure S1.Fingerprint region of the NOESY spectrum of 8mer DNA showing the aromatic−H1′ intranucleotide and sequential connectivities at 283K.All labels are shown as the respective intranucleotide aromatic-1' crosspeak for the indicated nucleotide.The black lines represent the strand containing the mismatched T. The blue lines represent the strand containing the base-paired G.The δH values have been referenced to the temperature-dependent HDO signal per Gottlieb et al.

Figure S5 .
Figure S5.Fingerprint region of the NOESY spectrum of 8mer CpG:T DNA showing the aromatic−H1′ intranucleotide and sequential connectivities at 283K.All labels are shown above the respective intranucleotide aromatic-1' crosspeak for the indicated nucleotide.The black lines represent the strand containing the mismatched T. The blue lines represent the strand containing the base-paired G.The δH values have been referenced to the temperature-dependent HDO signal per Gottlieb et al.

Figure S9 .Figure S11 .
Figure S9.Fingerprint region of the NOESY spectrum of 8mer TpG:T DNA showing the aromatic−H1′ intranucleotide and sequential connectivities at 283K.All labels are shown above the respective intranucleotide aromatic-1' crosspeak for the indicated nucleotide.The black lines represent the strand containing the mismatched T. The blue lines represent the strand containing the base-paired G.The δH values have been referenced to the temperature-dependent HDO signal per Gottlieb et al.

Figure S12 .
Figure S12.Fingerprint region of the NOESY spectrum of 8mer GpG:T DNA showing the aromatic−H1′ intranucleotide and sequential connectivities at 283K.All labels are shown above the respective intranucleotide aromatic-1' crosspeak for the indicated nucleotide.The black lines represent the strand containing the mismatched T. The blue lines represent the strand containing the base-paired G.The δH values have been referenced to the temperature-dependent HDO signal per Gottlieb et al.

Figure S16 .Figure S19 .
Figure S16.Fingerprint region of the NOESY spectrum of 8mer ApG:T DNA showing the aromatic−H1′ intranucleotide and sequential connectivities at 283K.All labels are shown above the respective intranucleotide aromatic-1' crosspeak for the indicated nucleotide.The black lines represent the strand containing the mismatched T. The blue lines represent the strand containing the base-paired G.The δH values have been referenced to the temperature-dependent HDO signal per Gottlieb et al.

Table S1 .
1 H Assignments for 8mer DNA at 283K.The δH values have been referenced to the temperature-dependent HDO signal per Gottlieb et al.

Table S2 .
31P assignments for 8mer control DNA as a function of temperature.The δP values have been externally referenced to H3PO4 at 0.00 ppm via coaxial insert.

Table S4 .
1 H Assignments for 8mer CpG:T DNA at 288K.The δH values have been referenced to the temperature-dependent HDO signal per Gottlieb et al.

Table S5 .
31P assignments for 8mer CpG:T control DNA as a function of temperature.The δP values have been externally referenced to H3PO4 at 0.00 ppm via coaxial insert.

Table S6 .
1 H Assignments for 8mer TpG:T DNA at 288K.The δH values have been referenced to the temperature-dependent HDO signal per Gottlieb et al.

Table S7 .
31P assignments for 8mer TpG:T control DNA as a function of temperature.The δP values have been externally referenced to H3PO4 at 0.00 ppm via coaxial insert.

Table S8 .
1 H Assignments for 8mer GpG:T DNA at 288K.The δH values have been referenced to the temperature-dependent HDO signal per Gottlieb et al.

Table S9 .
31P assignments for 8mer GpG:T control DNA as a function of temperature.The δP values have been externally referenced to H3PO4 at 0.00 ppm via coaxial insert.

Table S10 .
The δH values have been referenced to the temperature-dependent HDO signal per Gottlieb et al.Note that some protons were unable to be assigned unambiguously.
1 H Assignments for 8mer ApG:T DNA at 278K.

Table S11 .
31P assignments for 8mer ApG:T control DNA as a function of temperature.The δP values have been externally referenced to H3PO4 at 0.00 ppm via coaxial insert.