Probing the effects of DNA-protein interactions on DNA hole transport: the N-terminal histone tails modulate the distribution of oxidative damage and chemical lesions in the nucleosome core particle

The ability of DNA to transport positive charges, or holes, over long distances is well-established, but the mechanistic details of how this process is influenced by packaging into DNA-protein complexes have not been fully delineated. In eukaryotes, genomic DNA is packaged into chromatin through its association with the core histone octamer to form the nucleosome core particle (NCP), a complex whose structure can be modulated through changes in the local environment and the histone proteins. Because (i) varying the salt concentration and removing the histone tails influence the structure of the NCP in known ways and (ii) previous studies have shown that DNA hole transport (HT) occurs in the nucleosome, we have used our previously described 601 sequence NCPs to test the hypothesis that DNA HT dynamics can be modulated by structural changes in a DNA-protein complex. We show that at low salt concentrations there is a sharp increase in long-range DNA HT efficiency in the NCP as compared to naked DNA. This enhancement of HT can be negated by either removal of the histone tails at low salt concentrations or disruption of the interaction of the packaged DNA and the histone tails by increasing the buffer's ionic strength. Association of the histone tails with 601 DNA at low salt concentrations shifts the guanine damage spectrum to favor lesions like 8-oxoguanine in the NCP, most likely through modulation of the rate of the reaction of the guanine radical cation with oxygen. These experimental results indicate that for most genomic DNA, the influence of DNA-protein interactions on DNA HT will depend strongly on the level of protection of the DNA nucleobases from oxygen. Further, these results suggest that the oxidative damage arising from DNA HT may vary in different genomic regions depending on the presence of either euchromatin or heterochromatin.     

Computer modeling demonstrates that electrostatic attraction of nucleosomal DNA is mediated by histone tails

We conducted molecular dynamics computer simulations of charged histone tail-DNA interactions in systems mimicking nucleosome core particles (NCP) . In a coarse-grained model, the NCP is modeled as a negatively charged spherical particle with flexible polycationic histone tails attached to it in a dielectric continuum with explicit mobile counterions and added salt. The size, charge, and distribution of the tails relative to the core were built to mimick real NCP. In this way, we incorporate attractive ion-ion correlation effects due to fluctuations in the ion cloud and the attractive entropic and energetic tail-bridging effects. In agreement with experimental data, increase of monovalent salt content from salt-free to physiological concentration leads to the formation of NCP aggregates; likewise, in the presence of MgCl₂, the NCPs form condensed systems via histone-tail bridging and accumulation of counterions. More detailed mechanisms of the histone tail-DNA interactions and dynamics have been obtained from all-atom molecular dynamics simulations (including water), comprising three DNA 22-mers and 14 short fragments of the H4 histone tail (amino acids 5–12) carrying three positive charges on lysine⁺ interacting with DNA. We found correlation of the DNA-DNA distance with the presence and association of the histone tail between the DNA molecules.     

Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 Å crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location −5.0 to −2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA–DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn²⁺ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.     

H2A and H2B tails are essential to properly reconstitute nucleosome core particles

The conformation of recombinant Nucleosome Core Particles (NCPs) lacking H2A and H2B histone tails (gH2AgH2B) are studied. The migration of these particles in acrylamide native gels is slowed down compared to intact reconstituted NCPs. gH2AgH2B NCPs are also much more sensitive to nuclease digestion than intact NCPs. Small angle X-ray scattering (SAXS) experiments point out that the absence of H2A and H2B tails produces small but significant conformational changes of the octamers conformation (without wrapped DNA), whereas gH2AgH2B NCP conformations are significantly altered. A separation of about 25–30 bp from the core could account for the experimental curves, but other types of DNA superhelix deformation cannot be excluded. The distorted gH2AgH2B octamer may not allow the correct winding of DNA around the core. The absence of the H2A and H2B tails would further prevent the secondary sliding of the DNA around the core and therefore impedes the stabilisation of the particle. Cryo-electron microscopy on the same particles also shows a detachment of DNA portions from the particle core. The effect is even stronger because the vitrification of the samples worsens the instability of gH2AgH2B NCPs.     

Transport of nucleosome core particles in semidilute DNA solutions

We studied the diffusion of native and trypsinized nucleosome core particles (NCPs), in aqueous solution and in concentrated DNA solutions (0.25-100 mg/ml) using fluorescence correlation spectroscopy (FCS). The highest DNA concentrations studied mimic the DNA density inside the cell nucleus. The diffusion coefficient of freely diffusing NCPs depends on the presence or absence of histone tails and is affected by the salt concentration due to the relaxation effect of counterions. NCPs placed in a network of long DNA molecules (30-50 kbp) reveal anomalous diffusion. We demonstrate that NCPs diffusion is in agreement with known particle transport in entangled macromolecular solutions as long as the histone tails are folded onto the particles. In contrast, when these tails are unfolded, the reversible adsorption of NCPs onto the DNA network has to be taken into account. This is confirmed by the fact that removal of the tails leads to reduction of the interaction between NCPs and the DNA network. The findings suggest that histone tail bridging plays an important role in chromatin dynamics.     

RAD51AP1 mediates RAD51 activity through nucleosome interaction

RAD51-associated protein 1 (RAD51AP1) is a key protein in the homologous recombination (HR) DNA repair pathway. Loss of RAD51AP1 leads to defective HR, genome instability, and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of donor DNA, synaptic complex assembly, and displacement-loop formation when tested with nucleosome-free DNA substrates. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. In this study, we show that RAD51AP1 binds to nucleosome core particles (NCPs), the minimum basic unit of chromatin in which approximately two superhelical turns of 147 bp double-stranded DNA are wrapped around one histone octamer with no free DNA ends remaining. We identified a C-terminal region in RAD51AP1, including its previously mapped DNA-binding domain, as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. Using in vitro surrogate assays of HR activity, we show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists in the RAD51-mediated search for donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.     

Energetics and affinity of the histone octamer for defined DNA sequences

Previous studies have compared the relative free energies for histone octamer binding to various DNA sequences; however, no reports of the equilibrium binding affinity of the octamer for unique sequences have been presented. It has been shown that nucleosome core particles (NCPs) dissociate into free DNA and histone octamers (or free histones) on dilution without generation of stable intermediates. Dissociation is reversible, and an equilibrium distribution of NCPs and DNA is rapidly attained. Under low ionic strength conditions (<400 mM NaCl), NCP dissociation obeys the law of mass action, making it possible to calculate apparent equilibrium dissociation constants (K(d)s) for NCPs reconstituted on defined DNA sequences. We have used two DNA sequences that have previously served as model systems for nucleosome reconstitution studies, human alpha-satellite DNA and Lytechinus variegatus 5S DNA, and find that the octamer exhibits K(d)s of 0.03 and 0.06 nM, respectively, for these sequences at 50 mM NaCl. These DNAs form NCPs that are approximately 2 kcal/mol more stable than total NCPs isolated from cellular chromatin. As for mixed-sequence NCPs, increasing ionic strength or temperature promotes dissociation. van't Hoff plots of K(a)s versus temperature reveal that the difference in binding free energy for alpha-satellite and 5S NCPs compared to bulk NCPs is due almost entirely to a more favorable entropic component for NCPs formed on the unique sequences compared to mixed-sequence NCPs. Additionally, we address the contribution of the amino-terminal tail domains of histones H3 and H4 to octamer affinity through the use of recombinant tailless histones.     

Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability

We engineered nucleosome core particles (NCPs) with two site-specific cysteine crosslinks that increase the stability of the particle. The first disulfide was introduced between the two copies of H2A via an H2A-N38C point mutation, effectively crosslinking the two H2A/H2B heterodimers together to stabilize the histone octamer against H2A/H2B dimer dissociation. The second crosslink was engineered between an R40C point mutation on the N-terminal tail of H3 and the NCP DNA ends by the introduction of a convertible nucleotide. This crosslink maintains the nucleosome DNA in a fixed translational setting relative to the histone octamer and prevents dilution-driven dissociation. The X-ray crystal structures of NCPs containing the disulfides in isolation and in combination were determined. Both disulfides stabilize the structure of the NCP without disturbing the overall structure. Nucleosomes containing these modifications will be advantageous for biochemical and structural studies as a consequence of their greater resistance to dissociation during high dilution in purification, elevated salt for crystallization and vitrification for cryogenic electron microscopy.     

Crystal Structures of Nucleosome Core Particles Containing the ‘601’ Strong Positioning Sequence

Nucleosome positioning plays a key role in genomic regulation by defining histone-DNA context and by modulating access to specific sites. Moreover, the histone-DNA register influences the double-helix structure, which in turn can affect the association of small molecules and protein factors. Analysis of genomic and synthetic DNA has revealed sequence motifs that direct nucleosome positioning in vitro; thus, establishing the basis for the DNA sequence dependence of positioning would shed light on the mechanics of the double helix and its contribution to chromatin structure in vivo. However, acquisition of well-diffracting nucleosome core particle (NCP) crystals is extremely dependent on the DNA fragment used for assembly, and all previous NCP crystal structures have been based on human α-satellite sequences. Here, we describe the crystal structures of Xenopus NCPs containing one of the strongest known histone octamer binding and positioning sequences, the so-called ‘601’ DNA.Two distinct 145-bp 601 crystal forms display the same histone-DNA register, which coincides with the occurrence of DNA stretching-overtwisting in both halves of the particle around five double-helical turns from the nucleosome center, giving the DNA an ‘effective length’ of 147 bp. As we have found previously with stretching around two turns from the nucleosome center for a centromere-based sequence, the terminal stretching observed in the 601 constructs is associated with extreme kinking into the minor groove at purine-purine (pyrimidine-pyrimidine) dinucleotide steps. In other contexts, these step types display an overall nonflexible behavior, which raises the possibility that DNA stretching in the nucleosome or extreme distortions in general have unique sequence dependency characteristics. Our findings indicate that DNA stretching is an intrinsically predisposed site-specific property of the nucleosome and suggest how NCP crystal structures with diverse DNA sequences can be obtained.     

Influence of histone tails and H4 tail acetylations on nucleosome-nucleosome interactions

Nucleosome–nucleosome interaction plays a fundamental role in chromatin folding and self-association. The cation-induced condensation of nucleosome core particles (NCPs) displays properties similar to those of chromatin fibers, with important contributions from the N-terminal histone tails. We study the self-association induced by addition of cations [Mg²⁺, Ca²⁺, cobalt(III)hexammine³⁺, spermidine³⁺ and spermine⁴⁺] for NCPs reconstituted with wild-type unmodified histones and with globular tailless histones and for NCPs with the H4 histone tail having lysine (K) acetylations or lysine-to-glutamine mutations at positions K5, K8, K12 and K16. In addition, the histone construct with the single H4K16 acetylation was investigated. Acetylated histones were prepared by a semisynthetic native chemical ligation method. The aggregation behavior of NCPs shows a general cation-dependent behavior similar to that of the self-association of nucleosome arrays. Unlike nucleosome array self-association, NCP aggregation is sensitive to position and nature of the H4 tail modification. The tetra-acetylation in the H4 tail significantly weakens the nucleosome–nucleosome interaction, while the H4 K → Q tetra-mutation displays a more modest effect. The single H4K16 acetylation also weakens the self-association of NCPs, which reflects the specific role of H4K16 in the nucleosome–nucleosome stacking. Tailless NCPs can aggregate in the presence of oligocations, which indicates that attraction also occurs by tail-independent nucleosome–nucleosome stacking and DNA–DNA attraction in the presence of cations. The experimental data were compared with the results of coarse-grained computer modeling for NCP solutions with explicit presence of mobile ions.     

Enzymatic excision of uracil residues in nucleosomes depends on the local DNA structure and dynamics

The excision of uracil bases from DNA is accomplished by the enzyme uracil DNA glycosylase (UNG). Recognition of uracil bases in free DNA is facilitated by uracil base pair dynamics, but it is not known whether this same mechanistic feature is relevant for detection and excision of uracil residues embedded in nucleosomes. Here we investigate this question using nucleosome core particles (NCPs) generated from Xenopus laevis histones and the high-affinity "Widom 601" positioning sequence. The reactivity of uracil residues in NCPs under steady-state multiple-turnover conditions was generally decreased compared to that of free 601 DNA, mostly because of anticipated steric effects of histones. However, some sites in NCPs had equal or even greater reactivity than free DNA, and the observed reactivities were not readily explained by simple steric considerations or by global DNA unwrapping models for nucleosome invasion. In particular, some reactive uracils were found in occluded positions, while some unreactive uracils were found in exposed positions. One feature of many exposed reactive sites is a wide DNA minor groove, which allows penetration of a key active site loop of the enzyme. In single-turnover kinetic measurements, multiphasic reaction kinetics were observed for several uracil sites, where each kinetic transient was independent of the UNG concentration. These kinetic measurements, and supporting structural analyses, support a mechanism in which some uracils are transiently exposed to UNG by local, rate-limiting nucleosome conformational dynamics, followed by rapid trapping of the exposed state by the enzyme. We present structural models and plausible reaction mechanisms for the reaction of UNG at three distinct uracil sites in the NCP.     

Redefinition of the cleavage sites of DNase I on the nucleosome core particle

DNase I has been widely used for the footprinting of DNA-protein interactions including analyses of nucleosome core particle (NCP) structure. Our understanding of the relationship between the footprint and the structure of the nucleosome complex comes mainly from digestion studies of NCPs, since they have a well-defined quasi-symmetrical structure and have been widely investigated. However, several recent results suggest that the established consensus of opinion regarding the mode of digestion of NCPs by DNase I may be based on erroneous interpretation of results concerning the relationship between the NCP ends and the dyad axis. Here, we have used reconstituted NCPs with defined ends, bulk NCPs prepared with micrococcal nuclease and molecular modelling to reassess the mode of DNase I digestion. Our results indicate that DNase I cuts the two strands of the nucleosomal DNA independently with an average stagger of 4 nt with the 3'-ends protruding. The previously accepted value of 2 nt stagger is explained by the finding that micrococcal nuclease produces NCPs not with flush ends, but with approximately 1 nt 5'-recessed ends. Furthermore we explain why the DNA stagger is an even and not an odd number of nucleotides. These results are important for studies using DNase I to probe nucleosome structure in complex with other proteins or any DNA-protein complex containing B-form DNA. We also determine the origin of the 10n +/- 5 nt periodicity found in the internucleosomal ladder of DNase I digests of chromatin from various species. The explanation of the 10n +/- 5 nt ladder may have implications for the structure of the 30 nm fibre.     

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.     

Solution structure and backbone dynamics of Mason-Pfizer monkey virus (MPMV) nucleocapsid protein.

Retroviral nucleocapsid proteins (NCPs) are CCHC-type zinc finger proteins that mediate virion RNA binding activities associated with retrovirus assembly and genomic RNA encapsidation. Mason-Pfizer monkey virus (MPMV), a type D retrovirus, encodes a 96-amino acid nucleocapsid protein, which contains two Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc fingers connected by an unusually long 15-amino acid linker. Homonuclear, two-dimensional sensitivity-enhanced 15N-1H, three-dimensional 15N-1H, and triple resonance NMR spectroscopy have been used to determine the solution structure and residue-specific backbone dynamics of the structured core domain of MPMV NCP containing residues 21-80. Structure calculations and spectral density mapping of N-H bond vector mobility reveal that MPMV NCP 21-80 is best described as two independently folded, rotationally uncorrelated globular domains connected by a seven-residue flexible linker consisting of residues 42-48. The N-terminal CCHC zinc finger domain (residues 24-37) appears to adopt a fold like that described previously for HIV-1 NCP; however, residues within this domain and the immediately adjacent linker region (residues 38-41) are characterized by extensive conformational averaging on the micros-ms time scale at 25 degrees C. In contrast to other NCPs, residues 49-77, which includes the C-terminal CCHC zinc-finger (residues 53-66), comprise a well-folded globular domain with the Val49-Pro-Gly-Leu52 sequence and C-terminal tail residues 67-77 characterized by amide proton exchange properties and 15N R1, R2, and (1H-15N) NOE values indistinguishable to residues in the core C-terminal finger. Twelve refined structural models of MPMV NCP residues 49-80 (pairwise backbone RMSD of 0.77 A) reveal that the side chains of the conserved Pro50 and Trp62 are in van der Waals contact with one another. Residues 70-73 in the C-terminal tail adopt a reverse turn-like structure. Ile77 is involved in extensive van der Waals contact with the core finger domain, while the side chains of Ser68 and Asn75 appear to form hydrogen bonds that stabilize the overall fold of this domain. These residues outside of the core finger structure are conserved in D-type and related retroviral NCPs, e.g., MMTV NCP, suggesting that the structure of MPMV NCP may be representative of this subclass of retroviral NCPs.     

Recognizing and remodeling the nucleosome

The X-ray structure of the nucleosome core particle (NCP) has been a major milestone in the structural biology of chromatin. Since, our understanding how NCPs interact with multiple partners has been extending from single chromatin-binding domains recognizing post-translational modifications (PTMs) in histone tails towards the recognition of higher-order chromatin structure by multi-subunit chromatin remodeling complexes. The current review summarizes recent progress in the structural biology of nucleosome-recognition from chromatin-binding domains to multi-protein remodeling complexes.     

Histone Chaperone-Mediated Nucleosome Assembly Process

A huge amount of information is stored in genomic DNA and this stored information resides inside the nucleus with the aid of chromosomal condensation factors. It has been reported that the repeat nucleosome core particle (NCP) consists of 147-bp of DNA and two copies of H2A, H2B, H3 and H4. Regulation of chromosomal structure is important to many processes inside the cell. In vivo, a group of histone chaperones facilitate and regulate nucleosome assembly. How NCPs are constructed with the aid of histone chaperones remains unclear. In this study, the histone chaperone-mediated nucleosome assembly process was investigated using single-molecule tethered particle motion (TPM) experiments. It was found that Asf1 is able to exert more influence than Nap1 and poly glutamate acid (PGA) on the nucleosome formation process, which highlights Asf1’s specific role in tetrasome formation. Thermodynamic parameters supported a model whereby energetically favored nucleosomal complexes compete with non-nucleosomal complexes. In addition, our kinetic findings propose the model that histone chaperones mediate nucleosome assembly along a path that leads to enthalpy-favored products with free histones as reaction substrates.