Molecular recognition of the nucleosomal "supergroove"

Abstract

Chromatin is the physiological substrate in all processes involving eukaryotic DNA. By organizing 147 base pairs of DNA into two tight superhelical coils, the nucleosome generates an architecture where DNA regions that are 80 base pairs apart on linear DNA are brought into close proximity, resulting in the formation of DNA "supergrooves." Here, we report the design of a hairpin polyamide dimer that targets one such supergroove. The 2-A crystal structure of the nucleosome-polyamide complex shows that the bivalent "clamp" effectively crosslinks the two gyres of the DNA superhelix, improves positioning of the DNA on the histone octamer, and stabilizes the nucleosome against dissociation. Our findings identify nucleosomal supergrooves as platforms for molecular recognition of condensed eukaryotic DNA. In vivo, supergrooves may foster synergistic protein-protein interactions by bringing two regulatory elements into juxtaposition. Because supergroove formation is independent of the translational position of the DNA on the histone octamer, accurate nucleosome positioning over regulatory elements is not required for supergroove participation in eukaryotic gene regulation.

Fig. 1.

Site-specific recognition of nucleosomal DNA by clamp PAs. (A) NCP146 structure (PDB ID code 1AOI, ref. 2) viewed with the superhelical axis along the z axis. The particle pseudo-two-fold dyad axis (φ) is shown for orientation. DNA (blue and white) and associated histone proteins (H2A, yellow; H2B, red; H3, blue; H4, green) are shown in sphere or surface representation. (B) Supergrooves in NCP146. Shown is a different view of NCP146 with the superhelical axis along the y axis. Color scheme is the same as in A. One of the DNA supergrooves is indicated by two asterisks. (C) Chemical structures of clamp PAs, PW12 to -14. (D) Hydrogen bonding model of PW12 to its target DNA site. Circles with dots represent lone pairs of N3 of purines and O2 of pyrimidines. Circles containing H represent the N2 hydrogen of guanine. Putative hydrogen bonds are illustrated by dotted lines.

Fig. 2.

Clamp binding in the NCP146–PW12 complex. Color code for histone proteins is as in Fig. 1. DNA is shown in green and white, PW12 in green or magenta. (A) Overview of NCP146–PW12 structure, orientation same as in Fig. 1B. (B) Stereoview of PW12 bound to its target DNA site. Omit density for the clamp is shown at 2 σ contour level. Chains 1 and 2 denote the two hairpin moieties. (C) The linker in the clamp is buried between the two gyres of superhelical DNA. A close-up surface representation of the NCP146–PW12 structure is shown.

Fig. 3.

Schematic illustration of the predicted effect of PA clamps on nucleosome dissociation. In the absence of ligand binding, nucleosome dissociation initiates with unraveling of the DNA ends, followed by dissociation of the (H2A-H2B) dimers, and finally by the dissociation of the (H3-H4)₂ tetramer. Binding clamp to the nucleosomes leads to the formation of a closed ≈80-bp DNA supercoil that prevents further disassembly of the nucleosomes.

Fig. 4.

Stabilization of NCP146 by the clamp. (A) Nondenaturing gel analysis. NCP146 was subjected to serial dilution in a dilution buffer that contained either no PA (Top), or 100 nM PA1 (Middle) or PW12 (Bottom), as indicated. NCP146 samples were then subjected to electrophoresis on a 6% nondenaturing polyacrylamide gel, and a PhosphorImager of the gel is shown. N, NCP146; D, 146-bp DNA; A * denotes the stability of the NCP146 upon dilution in the presence of PW12. The side panel shows a darker contrast of lane 8 in the Bottom. (B) Graphical analysis of the dissociation data shown in A. The fraction of DNA remaining in NCP146, relative to the 10-nM sample, is plotted vs. NCP146 concentration. •, no PA; ▪, PA1; ⋄, PW12.