Protein-facilitated base flipping in DNA by cytosine-5-methyltransferase

Abstract

DNA methylation, various DNA repair mechanisms, and possibly early events in the opening of DNA as required for transcription and replication are initiated by flipping of a DNA base out of the DNA double helix. The energetics and structural mechanism of base flipping in the presence of the DNA-processing enzyme, cytosine 5-methyltransferase from HhaI (M.HhaI), were obtained through molecular dynamics based upon free-energy calculations. Free-energy profiles for base flipping show that, when in the closed conformation, M.HhaI lowers the free-energy barrier to flipping by 17 kcalmol and stabilizes the fully flipped state. Flipping is shown to occur via the major groove of the DNA. Structural analysis indicates that flipping is facilitated by destabilization of the DNA double-helical structure and substitution of DNA base-pairing and base-stacking interactions with DNA-protein interactions. The fully flipped state is stabilized by DNA-protein interactions that are enhanced upon binding of coenzyme. This study represents an atomic detail description of the mechanism by which a protein facilitates specific structural distortion in DNA.

Figure 1

(A) DNA double helix central three bases (tribase) showing flipping of the target C base (red) from the DNA double-helical conformation (Right) to a flipped conformation (Left) via the major or minor grooves. Orphan guanine is shown in green. (B) Schematic diagram of the pseudodihedral used to describe the flipping process. The pseudodihedral is defined by the center of masses of (i) the target C (red, A), (ii) its sugar moiety (purple, B), (iii) the adjacent 3′ sugar moiety (blue, C), and (iv) the 3′ GC base atoms (green, D). Hydrogens were omitted for clarity. (C) Diagram relating the periodic pseudodihedral to structural changes, where the DNA double-helical state corresponds to 10°, the flipped state to ≈195°, minor groove flipping as increasing from 10° to 195°, and major groove flipping as decreasing from 10° through 0–360° to 195°. (D) Open-binary DNA-M.HhaI complex (Left) and the closed-ternary DNA-M.HhaI-SAH complex (Right). DNA (green), target C base (red spheres), orphan G base (green spheres), catalytic and recognition domains of M.HhaI (blue), active-site loop (yellow), Ser-87 (purple), Gln-237 (gold), and SAH (gray) are shown. Arrows indicating the a priori minor and major groove-flipping pathways are shown on the open-binary complex structure. (E) Free-energy surfaces for base flipping for DNA in aqueous solution (black), the open DNA-M.HhaI binary complex (red), the closed DNA-M.HhaI binary complex (blue), and the DNA-M.HhaI-SAH ternary complex (green). Free-energy profiles have been offset to 0 kcal/mol at 10° (WC base-paired state). Energy barriers at 40° (minor groove) and 315° (major groove) for DNA in aqueous solution are in agreement with previous studies (14). Molecular images were generated with vmd (26).

Figure 2

(A) Central three DNA base pairs at the DNA WC base-paired state (x = 10°, Fig. 1C) for DNA in aqueous solution, in the open-binary complex, in the closed-binary complex, and in the ternary complex. Target C base (red) and orphan G base (green) are indicated. The two images for each complex are the same structures rotated ≈90°. (B and C, Left) Protein residues that interact with the central DNA tribase for the open-binary (purple) and closed-ternary complexes (blue) in the WC paired state (x = 10°) (B) and at 50% of the major groove barrier (x = 340°) (C). Residues interacting in both complexes are indicated by the purple **; the interactions are shown as purple and blue dashed lines for the open-binary and ternary complexes, respectively. Black dashed lines indicate interactions that occur in both complexes. Hydrogen bonds are defined as acceptor-to-donor distances ≤4 Å. (B and C, Right) DNA tribase from the ternary complex at B (x = 10°) and C (x = 340°). Ser-87 (purple), Gln-237 (gold), and protein–DNA hydrogen bonds (blue dashed lines) are shown. Structures were the minimized average Cartesian coordinates from the selected windows of the free-energy profile.