Lysine‐containing DNA‐binding regions on the surface of the histone octamer in the nucleosome core particle

Abstract

The DNA bound on the surface of the histone octamer in the nucleosome core particle partially protects the &-amino side-chains of a subset of the lysine residues from reductive methylation. Most of the strongly protected lysines, which probably define the path of the DNA on the octamer surface, are in the globular ('structured') regions of the core histones rather than in the N-terminal or C-terminal 'tails'. Analysis of the protected peptides shows that the three strongest lysine-containing DNA-binding sites in the core histones contain the sequence -Lys/ Arg-Lys-Thr/Ser-. On the assumption that the lysine-containing regions protected from chemical modification are also those found in lysine-DNA cross-links in another study [Mirzabekov et al. (1978) Proc. Nut1 Acad. Sci. USA 75, 41 84 -41 881, particular DNA-protected peptides may be tentatively assigned to particular DNA contact points. T h s leads to a more detailed description of the DNA-binding regions on the octamer surface in the nucleosome core particle. Strong contacts, reflected in strongly protected lysines, may well contribute to the distortion of the DNA from smooth bending [Richmond et al. (1984) Nature (Lond.) 311, 532-5371.A low-resolution (2.0 nm) structure, deduced for the nucleosome core particle, defined in outline the region within the structure occupied by particular histones, and the path of the DNA [1]. Subsequently these features were determined directly from X-ray diffraction analysis of nucleosome core particle crystals at 0.7 nm resolution [2]. However, the details of histone chain folding and of histone-DNA interactions remain at present unresolved. Some interactions may be particularly strong and may be responsible for distortion of the DNA from otherwise smooth bending [2]. They seem likely to involve electrostatic linkages between lysine and arginine side-chains on the surface of the histone octamer and DNA phosphates.We describe here a procedure for specifically radiolabelling lysine residues that are afforded protection against chemical modification in the native structure by the DNA. These are presumed to be engaged in electrostatic interaction with DNA phosphates and to direct the path of the DNA on the octarner surface. MATERIALS AND METHODSPreparation of nucleosome core particles and 'tail-less' core particles Chromatin was prepared from chicken erythrocyte nuclei [4] at AZ60 = 100 (read in 1 M NaOH) by digestion with micrococcal nuclease (5 units/ml nuclei for 4.25 min at 37°C) NazEDTA, pH 7.5. H1 and H5 were removed by treatment with the cation-exchange resin, AG5OW-X2 [6] and the stripped chromatin was dialysed into 10 mM Tris/HCl, 0.1 mM Na2EDTA, 0.25 mM phenylmethylsulphonyl fluoride (PMSF), pH 7.5, concentrated against solid sucrose and redialysed against the same buffer. It was digested to 146-basepair nucleosome core particles with micrococcal nuclease, under conditions determined from an analytical time course of digestion and analysis of the extracted DNA in a 6% polyacrylamide/7 M urea gel. The bulk digest wa...

A Novel Histone H4 Mutant Defective in Nuclear Division and Mitotic Chromosome Transmission

“…The lethal T82I substitution resides within the short beta-sheet region between helices 2 and 3 of the H4 histone fold (2). There is strong biochemical evidence from cross-linking and chemical protection experiments that this short beta-sheet region forms a DNA-binding surface in the nucleosome (6,44). In addition, recent molecular modeling studies indicate that this short beta-sheet region in histone H4 is one of the ''paired-element motifs'' that form the DNA-binding surfaces of the histone octamer (2,3).…”

Rearrangement of the histone H2A C-terminal domain in the nucleosome.

“…Reductive methylation has shown that both the C-terminal end and the globular domains of histone H2A are strongly bound to core particle DNA (26 (10,19). Stars mark the histone H2A contact at the dyad axis (nt 77).…”

“…2). The weak remaining signal is probably due to the crosslinking of Lys"8, which is situated in the conserved and strongly DNA-bound (L)PKK motif ofthe H2A C-terminal domain (26) (Fig. 2).…”

“…In the present study, using zero-length covalent histone-DNA crosslinking, we demonstrate that in isolated core particles the flexible trypsin-sensitive C-terminal domain of histone H2A is bound to the dyad axis, whereas the globular domain is bound to the end ofthe nucleosomal DNA. By taking into consideration the results of our previous histone-DNA crosslinking experiments (10,29,31) and the results of other investigators (26,30,32,34), we discuss the possible arrangement of the histone H2A C-terminal domain in chromatin and in intact nuclei and its rearrangement after the removal of linker DNA.…”

Epigenetic Inheritance of Centromeres

“…However, this argument overlooks the fact that the classical crosslinking reageant that we used, dimethylsuberimidate, was chosen because it has an 11Å linker and is known to crosslink only H2A, H2B, and H4 lysines far away from one another across the solvent-accessible surface of the H3 nucleosome (Kornberg and Thomas 1974;Suda and Iwai 1979). In any case, the lysines in the H3 dimerization domain are evidently not solvent accessible in the nucleosome core particle (Lambert and Thomas 1986;Davey et al 2002). This criticism also overlooked the fact that we were indeed able to cross-link CenH3 octamers reconstituted in vitro, although we never observed these forms in native CenH3 chromatin from nuclei (Dalal et al 2007b).…”