Experimental detection of conformational transitions between forms of DNA: problems and prospects.

Under different conditions, the DNA double helix can take different geometric forms. Of the large number of its conformations, in addition to the "canonical" B form, the A, C, and Z forms are widely known, and the D, Hoogsteen, and X forms are less known. DNA locally takes the A, C, and Z forms in the cell, in complexes with proteins. We compare different methods for detecting non-canonical DNA conformations: X-ray, IR, and Raman spectroscopy, linear and circular dichroism in both the infrared and ultraviolet regions, as well as NMR (measurement of chemical shifts and their anisotropy, scalar and residual dipolar couplings and inter-proton distances from NOESY (nuclear Overhauser effect spectroscopy) data). We discuss the difficulties in applying these methods, the problems of theoretical interpretation of the experimental results, and the prospects for reliable identification of non-canonical DNA conformations.

C-B-A Test of DNA Force Fields.

The DNA duplex may be locally strongly bent in complexes with proteins, for example, with polymerases or in a nucleosome. At such bends, the DNA helix is locally in the noncanonical forms A (with a narrow major groove and a large amount of north sugars) or C (with a narrow minor groove and a large share of BII phosphates). To model the formation of such complexes by molecular dynamics methods, the force field is required to reproduce these conformational transitions for a naked DNA. We analyzed the available experimental data on the B-C and B-A transitions under the conditions easily implemented in modeling: in an aqueous NaCl solution. We selected six DNA duplexes which conformations at different salt concentrations are known reliably enough. At low salt concentrations, poly(GC) and poly(A) are in the B-form, classical and slightly shifted to the A-form, respectively. The duplexes ATAT and GGTATACC have a strong and salt concentration dependent bias toward the A-form. The polymers poly(AC) and poly(G) take the C- and A-forms, respectively, at high salt concentrations. The reproduction of the behavior of these oligomers can serve as a test for the balance of interactions between the base stacking and the conformational flexibility of the sugar-phosphate backbone in a DNA force field. We tested the AMBER bsc1 and CHARMM36 force fields and their hybrids, and we failed to reproduce the experiment. In all the force fields, the salt concentration dependence is very weak. The known B-philicity of the AMBER force field proved to result from the B-philicity of its excessively strong base stacking. In the CHARMM force field, the B-form is a result of a fragile balance between the A-philic base stacking (especially for G:C pairs) and the C-philic backbone. Finally, we analyzed some recent simulations of the LacI-, SOX-4-, and Sac7d-DNA complex formation in the framework of the AMBER force field.

Variability of the DNA Backbone Geometry in DNA-Protein Complexes: Experimental Data Analysis.

We have analyzed and compared the available experimental data (PDB) on the backbone geometry of the DNA in solution (NMR), in crystals (X-rays), and in complexes with proteins (X-rays and cryo-electron microscopy). The deoxyribose (pseudorotational angle τ0) and ε/ζ (BI-BII transition in phosphates) flexibilities are practically the same in the four samples. The α/γ mobility is minimal in crystalline DNA: on the histograms, there is one canonical and one noncanonical t/t peak. The α/γ mobility increases in DNA solutions (three more noncanonical peaks) and is maximal in DNA-protein complexes (another additional peak). On a large amount of data, we have confirmed that the three main degrees of freedom of the sugar-phosphate backbone are "orthogonal": changes in any of the angles τ0, (ζ-ε), and (γ-α) occur, as a rule, at a constant (usually canonical) value of any other. In the DNA-protein complexes, none of the geometrical parameters commonly used to distinguish the A and B forms of DNA, except for Zp and its simpler analog Zp', show an unambiguous correlation with τ0. Proteins, binding to DNA, in 59% of cases change the local shape of the helix up to the characteristic of the A-form without switching the deoxyribose conformation from south to north. However, we have found simple local characteristics of one nucleotide that correlate with the angles τ0 and (ζ-ε). These are the angles C3'C1'N* and C4'C3'P(2), respectively. They are orthogonal in DNA-protein complexes exactly as the pair τ0 and (ζ-ε). Most characteristics of DNA in complexes with proteins are the same in X-ray and in cryo-EM data, except for the histogram for the angle τ0. We offer a possible explanation for this difference. We also discuss the artifacts on the ε/ζ histogram for DNA in solutions caused by the currently used NMR refinement protocols.

Kinetics of the Conformational Transformation between B- and A-Forms in the Drew-Dickerson Dodecamer.

Some DNA sequences in crystals and in complexes with proteins can exist in the forms intermediate between the B- and A-DNA. Based on this, it was implied that the B-to-A transition for any DNA molecule should go through these intermediate forms also in kinetics. More precisely, the helix parameter Slide has to change first, and the molecule should take the E-form. After that, the Roll parameter changes. In the present work, we simulated the kinetics of the B-A transition in the Drew-Dickerson dodecamer, a known B-philic DNA oligomer. We used the "sugar" coarse-grained model that reproduces ribose flexibility, preserves sequence specificity, employs implicit water and explicit ions, and offers the possibility to vary friction. As the control parameter of the transition, we chose the volume available for a counterion and considered the change from a large to a small volume. In the described system, the B-to-A conformational transformation proved to correspond to a first-order phase transition. The molecule behaves like a small cluster in the region of such a transition, jumping between the A- and B-forms in a wide range of available volumes. The viscosity of the solvent does not affect the midpoint of the transition but only the overall mobility of the system. All helix parameters change synchronously on average, we have not observed the sequence "Slide first, Roll later" in kinetics, and the E-DNA is not a necessary step for the transition between the B- and A-forms in the studied system. So, the existence of the intermediate DNA forms requires specific conditions, shifting the common balance of interactions: certain nucleotide sequence in specific solution or/and the interaction with some protein.