Base excision repair in nucleosomes lacking histone tails

Recently, we developed an in vitro system using human uracil DNA glycosylase (UDG), AP endonuclease (APE), DNA polymerase beta (pol beta) and rotationally positioned DNA containing a single uracil associated with a 'designed' nucleosome, to test short-patch base excision repair (BER) in chromatin. We found that UDG and APE carry out their catalytic activities with reduced efficiency on nucleosome substrates, showing a distinction between uracil facing 'out' or 'in' from the histone surface, while DNA polymerase beta (pol beta) is completely inhibited by nucleosome formation. In this report, we tested the inhibition of BER enzymes by the N-terminal 'tails' of core histones that take part in both inter- and intra-nucleosome interactions, and contain sites of post-translational modifications. Histone tails were removed by limited trypsin digestion of 'donor' nucleosome core particles and histone octamers were exchanged onto a nucleosome-positioning DNA sequence containing a single G:U mismatch. The data indicate that UDG and APE activities are not significantly enhanced with tailless nucleosomes, and the distinction between rotational settings of uracil on the histone surface is unaffected. More importantly, the inhibition of pol beta activity is not relieved by removal of the histone tails, even though these tails interact with DNA in the G:U mismatch region. Finally, inclusion of X-ray cross complement group protein 1 (XRCC1) or Werner syndrome protein (WRN) had no effect on the BER reactions. Thus, additional activities may be required in cells for efficient BER of at least some structural domains in chromatin.     

Differential dissociation of histone tails from core chromatin

The dissociation of the trypsin-sensitive basic tails of the core histones in core chromatin has been followed as a function of [NaCl] using proton NMR spectroscopy. The tails dissociate in a highly cooperative all or none manner over the salt concentration range 0.2-0.6 M, that is, below the salt concentration required to dissociate the complete molecule. Assuming that each basic tail dissociates independently, the total number of salt linkages involved in binding the tails to DNA is 103. This equals the number of basic side chains in the tails of an octamer. The standard free energy of dissociation, delta G degree, in 1 M NaCl at 297 K is 3.6 kcal/mol. Temperature had no effect on the extent of dissociation up to 45 degrees C. However, between 45 and 65 degrees C, where the premelting transition in the core chromatin occurs, the tails dissociated completely. Dissociation of the tails was associated with a conformational transition in the DNA consistent with loss of supercoiling. From this, and the results of a previous study, it can be shown that the structured, trypsin-resistant domain of each core histone octamer makes 100 salt linkages to DNA. Thus, in 10 mM salt, each core octamer makes a total of 203 salt linkages to DNA.     

Role of histone tails in structural stability of the nucleosome

Histone tails play an important role in nucleosome structure and dynamics. Here we investigate the effect of truncation of histone tails H3, H4, H2A and H2B on nucleosome structure with 100 ns all-atom molecular dynamics simulations. Tail domains of H3 and H2B show propensity of α-helics formation during the intact nucleosome simulation. On truncation of H4 or H2B tails no structural change occurs in histones. However, H3 or H2A tail truncation results in structural alterations in the histone core domain, and in both the cases the structural change occurs in the H2Aα3 domain. We also find that the contacts between the histone H2A C terminal docking domain and surrounding residues are destabilized upon H3 tail truncation. The relation between the present observations and corresponding experiments is discussed.     

More than just tails: intrinsic disorder in histone proteins

Many biologically active proteins are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions are very common in nature, abundantly found in all organisms, where they carry out important biological functions. The functions of these proteins complement the functional repertoire of "normal" ordered proteins, and many protein functional classes are heavily dependent on intrinsic disorder. Among these disorder-centric functions are interactions with nucleic acids and protein complex assembly. In this study, we present the results of comprehensive bioinformatics analyses of the abundance and roles of intrinsic disorder in 2007 histones from 746 species. We show that all the members of the histone family are intrinsically disordered proteins. Furthermore, intrinsic disorder is not only abundant in histones, but is absolutely necessary for various histone functions, starting from heterodimerization to formation of higher order oligomers, to interactions with DNA and other proteins, and to posttranslational modifications.     

Electrostatic interactions with histone tails may bend linker DNA in chromatin

Is linker DNA bent in the 30‐nm chromatin fiber at physiological conditions? We show here that electrostatic interactions between linker DNA and histone tails including salt condensation and release may bend linker DNA, thus affecting the higher order organization of chromatin. © 2005 Wiley Periodicals, Inc. Biopolymers 81: 20–28, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Complicated tails: histone modifications and the DNA damage response

In recent years, several ATP-dependent chromatin-remodeling complexes and covalent histone modifications have been implicated in the response to double-stranded DNA breaks (DSBs). When a DSB occurs, cells must identify the DSB, activate the DNA damage checkpoint, and repair the break. Chromatin modification appears to be important but not essential for each of these processes, yet its precise mechanistic roles are only beginning to come into focus. Here, we discuss the role of chromatin in signaling by the DNA damage checkpoint pathway.     

PARP1 ADP-ribosylates lysine residues of the core histone tails

The chromatin-associated enzyme PARP1 has previously been suggested to ADP-ribosylate histones, but the specific ADP-ribose acceptor sites have remained enigmatic. Here, we show that PARP1 covalently ADP-ribosylates the amino-terminal histone tails of all core histones. Using biochemical tools and novel electron transfer dissociation mass spectrometric protocols, we identify for the first time K13 of H2A, K30 of H2B, K27 and K37 of H3, as well as K16 of H4 as ADP-ribose acceptor sites. Multiple explicit water molecular dynamics simulations of the H4 tail peptide into the catalytic cleft of PARP1 indicate that two stable intermolecular salt bridges hold the peptide in an orientation that allows K16 ADP-ribosylation. Consistent with a functional cross-talk between ADP-ribosylation and other histone tail modifications, acetylation of H4K16 inhibits ADP-ribosylation by PARP1. Taken together, our computational and experimental results provide strong evidence that PARP1 modifies important regulatory lysines of the core histone tails.     

Mechanistic similarities in recognition of histone tails and DNA by epigenetic readers

The past two decades have witnessed rapid advances in the identification and characterization of epigenetic readers, capable of recognizing or reading post-translational modifications in histones. More recently, a new set of readers with the ability to interact with the nucleosome through concomitant binding to histones and DNA has emerged. In this review, we discuss mechanistic insights underlying bivalent histone and DNA recognition by newly characterized readers and highlight the importance of binding to DNA for their association with chromatin.     

Proteolytic clipping of histone tails: the emerging role of histone proteases in regulation of various biological processes

Chromatin is a dynamic DNA scaffold structure that responds to a variety of external and internal stimuli to regulate the fundamental biological processes. Majority of the cases chromatin dynamicity is exhibited through chemical modifications and physical changes between DNA and histones. These modifications are reversible and complex signaling pathways involving chromatin-modifying enzymes regulate the fluidity of chromatin. Fluidity of chromatin can also be impacted through irreversible change, proteolytic processing of histones which is a poorly understood phenomenon. In recent studies, histone proteolysis has been implicated as a regulatory process involved in the permanent removal of epigenetic marks from histones. Activities responsible for clipping of histone tails and their significance in various biological processes have been observed in several organisms. Here, we have reviewed the properties of some of the known histone proteases, analyzed their significance in biological processes and have provided future directions.     

Role of the histone "tails" in the folding of oligonucleosomes depleted of histone H1.

An oligonucleosome 12-mer was reconstituted in the absence of linker histones, onto a DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Ly-techinus variegatus (Simpson, R. T., Thoma, F. S., and Burbaker, J. M. (1985) Cell 42, 799-808). The ionic strength-dependent folding of this nucleohistone complex was compared with that of a native oligonucleosome fraction obtained from chicken erythrocyte chromatin, which had been carefully stripped of linker histones and fractionated in sucrose gradients. The DNA of this native fraction exhibited a narrow size distribution centered around the length of the 208-12 DNA template used in the reconstituted complex. These two complexes displayed very similar hydrodynamic behavior as judged by sedimentation velocity analysis. By combining these data with electron microscopy analysis, it was shown that the salt-dependent folding of oligonucleosomes in the absence of linker histones involves the bending of the linker DNA region connecting adjacent nucleosomes. It was also found that selective removal by trypsin of the N-terminal regions ("tails") of the core histones prevents the oligonucleosome chains from folding. Thus, in the absence of these histone domains, the bending of the linker DNA necessary to bring the nucleosomes in contact is completely abolished. In addition to the complete lack of folding, removal of the histone tails results in an unwinding at low salt of a 20-base pair region at each flanking side of the nucleosome core particle. The possible functional relevance of these results is discussed.     

Modifications of histone cores and tails in V(D)J recombination

The organization of chromatin and modifications to the tails of histone proteins are thought to be important in regulating the rearrangement of V, D and J gene segments, which encode immunoglobulins and T-cell receptors. A recent study shows that methylated lysine 79 in the core region of histone H3 also plays a role by providing a euchromatic 'mark' that may regulate access of the V(D)J recombinase.     

Role of histone tails in the conformation and interactions of nucleosome core particles

The goal of this work was to test the role of the histone tails in the emergence of attractive interactions between nucleosomes above a critical salt concentration that corresponds to the complete tail extension outside the nucleosome [Mangenot, S., et al (2002) Biophys. J. 82, 345-356; Mangenot, S., et al (2002) Eur. Phys. J. E 7, 221-231]. Small angle X-ray scattering experiments were performed in parallel with intact and trypsin tail-deleted nucleosomes with 146 +/- 3 bp DNA. We varied the monovalent salt concentration from 10 to 300 monovalent salt concentration and followed the evolution of (i) the second virial coefficient that characterizes the interactions between particles and (ii) the conformation of the particle. The attractive interactions do not emerge in the absence of the tails, which validates the proposed hypothesis.     

H3 and H4 histone tails play a central role in the interactions of recombinant NCPs

Using small-angle x-ray scattering, we probe the effect of histone tails on both internucleosomal interactions and nucleosome conformation. To get insight into the specific role of H3 and H4 histone tails, perfectly monodisperse recombinant nucleosome core particles were reconstituted, either intact or deprived of both H3 and H4 histone tails (gH3gH4). The main result is that H3 and H4 histone tails are necessary to induce attractive interactions between NCPs. A pair potential model was used to describe interactions between NCPs. At all salt concentrations, interactions between gH3gH4 NCPs are best described by repulsive interactions exclusively. For intact NCPs, an additional attractive term, with a 5–10 kT magnitude and 20 Å range, is required to account for interparticle interactions above 50 mM monovalent salt. Regarding conformation, intact NCPs in solution are similar to NCPs in 3D crystals. gH3gH4 NCPs instead give rise to slightly different small-angle x-ray scattering curves that can be understood as a more opened conformation of the particle, where DNA ends are slightly detached from the core.