5-Methylation of cytosine in CG:CG base-pair steps: a physicochemical mechanism for the epigenetic control of DNA nanomechanics

Abstract

van der Waals density functional theory is integrated with analysis of a non-redundant set of protein-DNA crystal structures from the Nucleic Acid Database to study the stacking energetics of CG:CG base-pair steps, specifically the role of cytosine 5-methylation. Principal component analysis of the steps reveals the dominant collective motions to correspond to a tensile "opening" mode and two shear "sliding" and "tearing" modes in the orthogonal plane. The stacking interactions of the methyl groups globally inhibit CG:CG step overtwisting while simultaneously softening the modes locally via potential energy modulations that create metastable states. Additionally, the indirect effects of the methyl groups on possible base-pair steps neighboring CG:CG are observed to be of comparable importance to their direct effects on CG:CG. The results have implications for the epigenetic control of DNA mechanics.

FIG. 1

Schematic illustration of important base-pair step parameters of canonical A, B and C forms of DNA. Also included are sample stacking diagrams for a CG:CG step, showing the exact spatial displacements of chemical units in fiber models. The lower rigid body in the upper schematics is denoted by the lightly shaded base pair in the lower stacking diagram. The shaded edges on the schematic blocks and the right edges of the stacking diagrams both correspond to the minor-groove edges of base pairs. The pink dashed lines represent hydrogen bonds. Schematics adapted from Reference and stacked CG step images computed with X3DNA.

FIG. 2

5-methylation of cytosine, an important epigenetic modification. The methyl group that replaces the hydrogen is circled in red. Green represents carbon, white hydrogen, red oxygen, and blue nitrogen. Graphics generated using PyMOL.

FIG. 3

The rigid-body configuration of a DNA base-pair step is specified by six local parameters per base pair and six step parameters for successive base-pair steps. There are three translational and three rotational degrees of freedom for each kind of rigid-body motion. Figure adapted with permission from Reference .

FIG. 4

The first principal component of CG:CG steps, a tensile ‘opening’ mode of the crack between DNA strands. From left to right, respectively, are images for steps that are five negative normal mode units from the mean, at the mean, and five positive units away. The upper and lower rows display views from the top-down and looking into the minor groove. The lower base pair is labelled by C1 bonded to G4, while the upper one is labelled by G2 and C3. The pink dashed lines represent hydrogen bonds. Molecular images created with 3DNA.

FIG. 5

The second principal component of CG:CG steps, a shear ‘sliding’ mode. See the caption of Figure 4 for explanation of notations and symbols.

FIG. 6

The third principal component of CG:CG steps, a shear ‘tearing’ mode. See the caption of Figure 4 for explanation of notations and symbols.

FIG. 7

Stacking energy landscapes for each of the three principal components, with and without methylation. The horizontal axes are labelled by the variation of twist along each of the modes.

FIG. 8

The individual contributions of one and two methyl groups to the effective stacking potential of the step, as measured by the energy difference, with respect to the unmethylated state, of calculations with one or both C5 groups methylated, respectively. The horizontal axes are labelled by the variation of twist along each of the modes.

FIG. 9

From left to right are minor-groove views of the low-twist regimes of the opening, sliding and tearing modes, respectively. As illustrated, in these regimes, the overlap area between C3 and G4 is greater. This leads to an enhancement in the stacking interactions of a methyl group at the C5 position with the adjacent guanine. The pink dashed lines represent hydrogen bonds

FIG. 10

As a base-pair step moves along the landscape of its conformational modes, the methyl group may be interpreted as having a set of moving ‘non-covalent’ dihedral angles with the atoms in the step, generated by the ‘angle’ that the 5-methylcytosine makes with the remainder of the base-pair step. In analogy to its more commonly discussed analog in covalent bonding, this torsional variation creates an effective torsional ‘potential’, which, like any periodic potential, can be decomposed into a superposition of harmonics of varying wavelength. In this schematic, A may be interpreted as a methyl group, the B–C line as the cytosine, and D as all other atoms in the step.

FIG. 11

Indirect stacking interaction energies of 5-methylcytosine with possible steps neighboring the CG:CG step. For AC:GT, GA:TC and GG:CC steps, there is only one possible cytosine that can connect to a CG:CG, and thus be potentially methylated. For GC:GC steps, however, it is possible for one or both of its cytosines to be methylated.