Enhanced C/EBP binding to G·T mismatches facilitates fixation of CpG mutations in cancer and adult stem cells

Abstract

Highlights d CpG core of C/EBP binding sites is prone to mutations in adult stem cells and cancers d CEBPB has enhanced affinity for binding sites with CpG$TpG mismatches d A model of fixation of mutations in C/EBP sites through enhanced C/EBP binding

Backbone Conformational Equilibrium in Mismatched DNA Correlates with Enzyme Activity

“…We analyzed five non-palindromic sequences both to examine sequence specificity and to remove any possible cross-strand effects from self-complementary duplexes. We used 8bp duplexes to reduce spectral overlap in the 31 P dimension, which remains a significant obstacle even in these short sequences. Throughout the manuscript when "mismatch" is used in the context of our data, we are referring to the T in the T:G base pair, as the G (referred to as the base-pairing partner) is assumed to be the correct nucleotide based upon the C:G context.…”

“…1D 31 P temperature study experiments were performed with a spectral window of 6000 Hz and a relaxation delay of 1−2 s, and 1024 scans were recorded between 5 and 25 °C for temperature studies with 5 °C increments. The 1D experiments were repeated with a coaxial insert containing 85% phosphoric acid used as an external reference set to 0.00 ppm for all 31 P peaks.…”

“…1D 31 P temperature study experiments were performed with a spectral window of 6000 Hz and a relaxation delay of 1−2 s, and 1024 scans were recorded between 5 and 25 °C for temperature studies with 5 °C increments. The 1D experiments were repeated with a coaxial insert containing 85% phosphoric acid used as an external reference set to 0.00 ppm for all 31 P peaks. 1 H-31 P HSQC experiments were performed with 2048 complex points for the t2 dimension, 256 t1 increments, and a relaxation delay of 1−2 s. The spectral width was 4000 Hz in f2 and 1950 Hz in f1.…”

“…As needed, spectra were compared to corresponding regions in the TOCSY, as is the established protocol for distinguishing through-space and through-bond correlations. 36−38 31 P isotropic chemical shifts (δP) were assigned (±0.02 ppm) by identifying correlations in 1 H- 31 P HSQC experiments as a function of the same temperature range as the 1D spectra (i.e., 5−25 °C). The primary correlations were between the phosphate peak for a dinucleotide pair (5′-XpY-3′) and the H4′ from the 3′ nucleotide in the pair.…”

“…has also been studied extensively 22,23 and other types of proteins such as CEBPβ have varying degrees of activity and binding strength on DNA containing T:G mismatches. 30,31 To attempt to quantify a relationship to enzyme properties, we have determined the equilibrium conformational populations of the DNA backbone using 31 P solution NMR. The specific conformation of the DNA phosphodiester backbone is described by a series of dihedral angles, 32,33 where the angles ε and ζ for the primary conformations are traditionally defined (Figure 1) as trans/g-in BI (ε -ζ ∼ −90°) and g-/trans in BII (ε -ζ ∼ +90°).…”