A molecular dynamics study of slow base flipping in DNA using conformational flooding

Abstract

Individual DNA bases are known to be able to flip out of the helical stack, providing enzymes with access to the genetic information otherwise hidden inside the helix. Consequently, base flipping is a necessary first step to many more complex biological processes such as DNA transcription or replication. Much remains unknown about this elementary step, despite a wealth of experimental and theoretical studies. From the theoretical point of view, the involved timescale of milliseconds or longer requires the use of enhanced sampling techniques. In contrast to previous theoretical studies employing umbrella sampling along a predefined flipping coordinate, this study attempts to induce flipping without prior knowledge of the pathway, using information from a molecular dynamics simulation of a B-DNA fragment and the conformational flooding method. The relevance to base flipping of the principal components of the simulation is assayed, and a combination of modes optimally related to the flipping of the base through either helical groove is derived for each of the two bases of the central guanine-cytosine basepair. By applying an artificial flooding potential along these collective coordinates, the flipping mechanism is accelerated to within the scope of molecular dynamics simulations. The associated free energy surface is found to feature local minima corresponding to partially flipped states, particularly relevant to flipping in isolated DNA; further transitions from these minima to the fully flipped conformation are accelerated by additional flooding potentials. The associated free energy profiles feature similar barrier heights for both bases and pathways; the flipped state beyond is a broad and rugged attraction basin, only a few kcal/mol higher in energy than the closed conformation. This result diverges from previous works but echoes some aspects of recent experimental findings, justifying the need for novel approaches to this difficult problem: this contribution represents a first step in this direction. Important structural factors involved in flipping, both local (sugar-phosphate backbone dihedral angles) and global (helical axis bend), are also identified.

FIGURE 1

Action of the flooding potential. The lower part shows the flooding potential V_fl, with different curves corresponding to different values of the flooding strength E_fl. The upper part shows the free energy landscape G along a generalized coordinate c_i (bold line), its local quasiharmonic approximation G^qh (bold dashes) and the flooded landscape once the potential has been applied (thin lines, different E_fl values). Inclusion of the flooding potential within the force field effectively lowers free energy barriers and accelerates conformational transitions.

FIGURE 2

Definition of the angle (31) used to quantify the flipping motion. The target cytosine is shown as solid sticks, its partner guanine as shaded sticks, and the two neighboring basepairs that define the local helical axis (perpendicular to the plane of the figure) as shaded lines. The flipping angle of the target base is symbolized by the thick open lines and arrow (refer to text for details).

FIGURE 3

Results of the PCA analysis on a set of configurations taken from a 20 ns MD simulation. Two subsets of atoms are considered: the DNA duplex without the capping basepairs, and the central basepair triplet. The two curves represent the cumulative amount of atomic motion retained by taking into account the first n PCA modes only. The bar plots denote the absolute value of the variation of the flipping angle along each eigenmode.

FIGURE 4

A flooding potential built upon the first five PCA eigenvectors, with E_fl = 35 kcal/mol, induces exaggerated bending of the DNA helix. Each point represents a snapshot from a flooding simulation and is associated with a global helical bend angle and a flooding potential value; these two parameters can be seen to exhibit opposite variations.

FIGURE 5

Synthetic results of 150 optimizations of the optimal flipping coordinate (see text), for cytosine (left) and guanine (right). The diamonds represent the average squared weight (over the 150 optimizations) of each PCA eigenvector within this coordinate (Eq. 13), the error bars the associated standard deviation. The corresponding PCA eigenvalues are shown as bars.

FIGURE 6

Contribution of the 20 first PCA eigenvectors to the four optimal generalized flipping coordinates (one per base and groove). The normalized weights of the eigenvectors in each case are shown as histograms (shaded bars for the minor groove pathway, solid for the major groove pathway).

FIGURE 7

Structures of the local free energy minima encountered along the flipping pathways. Refer to text for details.

FIGURE 8

Free energy profiles associated with the flipping of cytosine (top) and guanine (bottom) through each of the grooves, as a function of the flipping angle. Closed-form DNA conformations occur for values at ∼55° (dashed line). A positive (negative) variation of the angle denotes flipping through the major (minor) groove. The superimposed thick lines denote intact Watson and Crick hydrogen bonds between guanine and cytosine.

FIGURE 9

Variation of the χ dihedral angle during the flipping of the guanine and cytosine bases. The thick vertical line materializes the average closed-state conformation.

FIGURE 10

Values of relevant backbone dihedral angles as a function of the flipping angle. (a) ɛ, ζ, and γ dihedral angles of the flipping nucleoside; (b) ɛ, ζ, α, and γ of the nucleoside 5′ of the flipping base; (c) β, ɛ, and ζ of the deoxythymidine residue opposite the deoxyadenosine residue, 5′ of the flipping guanine; and (d) γ, ɛ, and ζ of the nucleoside 3′ of the flipping base. See text for details.

FIGURE 11

Global axis bending angle of the DNA helix as a function of the flipping angle. The thick vertical line materializes the average closed-state conformation.

FIGURE 12

Variation of the buckling, opening (°) and shear (A) parameters as a function of the flipping angle. The thick vertical line materializes the average closed-state conformation.

FIGURE 13

Free energy profile for the orientation of the flipped base with respect to the helical axis; at 0° the base plane is perpendicular to the helical axis.