³¹P NMR Investigation of Backbone Dynamics in DNA Binding Sites

Abstract

The backbone conformation of DNA plays an important role in the indirect readout mechanisms for protein-DNA recognition events. Thus, investigating the backbone dynamics of each step in DNA binding sequences provides useful information necessary for the characterization of these interactions. Here we use 31 P Dynamic NMR to characterize the backbone conformation and dynamics in the Dickerson Dodecamer, a sequence containing the EcoRI binding site, and confirm solid-state 2 H-NMR results showing that the C3pG4 and C9pG10 steps experience unique dynamics and that these dynamics are quenched upon cytosine methylation. In addition, we show that cytosine methylation affects the conformation and dynamics of neighboring nucleotide steps but this effect is localized to only near neighbors and base pairing partners. Lastly, we have been able to characterize the %BII in each backbone step and illustrate that the C3pG4 and C9pG10 favor the non-canonical BII conformation, even at low temperatures. Our results demonstrate that 31 P Dynamic NMR provides a robust and efficient method for characterizing the backbone dynamics in DNA. This allows simple, rapid determination of sequence-dependent dynamical information, providing a useful method for studying trends in protein-DNA recognition events.

Polarizable Force Field for DNA Based on the Classical Drude Oscillator: II. Microsecond Molecular Dynamics Simulations of Duplex DNA

“…Experimental data for the EcoRI sequence has been obtained using NMR, including refinement against SAXS ensembles. 81-82 Thus, it is possible to compare the backbone properties of the simulated EcoRI sequence with this important experimental observable. Figure 4 shows the fraction of BII as a function of the EcoRI sequence.…”

“…The NMR/SAXS data were taken from Schwieters and Clore, 81 who used NOE, RDC, J-coupling, and chemical shift anisotropy data in concert with SAXS spectra for refinement, using the results of the optimal N e = 4 model. The 31 P NMR data were taken from Tian et al 82 …”

Optimization of the CHARMM Additive Force Field for DNA: Improved Treatment of the BI/BII Conformational Equilibrium

“…The BII conformation was first characterized in a crystal structure 22 and subsequently observed using 31 P NMR chemical shifts and scalar coupling constants. 20,23,24 NMR data led to quantification of the intrinsic sequence-specific propensities to populate BII in solution for every DNA base-step. 25 Crucially, the BI/BII equilibrium affects the DNA helicoidal parameters, especially the twist, roll and base-pair displacement from the main helicoidal axis.…”

Sequence-Dependent DNA Flexibility Mediates DNase I Cleavage

“…43,44 The 31 P chemical shifts are very accurately measured in NMR; they can be expressed in terms of BI/BII ration. 40,45 Considering the phosphate group motion is of great interest because the BI ↔ BII equilibrium is intimately coupled to the deoxyribose conformational exchange 46,47 and to the DNA helicoidal parameters of twist, roll, slide, and basepair displacement (X-disp). 40,46,[48][49][50][51][52][53] The more a complementary dinucleotide favors the BII state, Fig.…”

“…Indeed, there is now strong evidence that sequence-dependent DNA backbone BI ↔ BII equilibrium plays a role in indirect readout by specific proteins through its effect on the overall shape of B-DNA. 44,45,50,[56][57][58][59][60][61] This study explores the relationship between DNase I cleavage efficiency and flexibility of free DNA on the basis of DNase I digestion patterns obtained on two well-characterized oligomers previously exhaustively studied by NMR. 40,41,60,62 The refined structures 50,60 show that the related helicoidal parameters vary significantly along the sequences, while remaining within the range ordinarily encountered in B-DNA; on average, these oligomers do not display sharp curvatures.…”