Oxidative charge transport through DNA in nucleosome core particles

Eukaryotic DNA is packaged into nucleosomes, made up of 146 bp of DNA wrapped around a core of histone proteins. We used photoexcited rhodium intercalators to explore DNA charge transport within these assemblies. Although histone proteins inhibit intercalation of the rhodium complex within the core particle, they do not prevent 5'-GG-3' oxidation, the signature of oxidative charge transport through DNA. Moreover, using rhodium intercalators tethered to the 5' terminus of the DNA, we found that guanine bases within the nucleosome can be oxidized from a distance of 24 bp. Histone binding did not affect the pattern and extent of this oxidation. Therefore, although the structure of the nucleosome core particle generally protects DNA from damage by solution-borne molecules, packaging within the nucleosome does not protect DNA from charge transfer damage through the base pair stack.     

Rigid assembly and Monte Carlo models of stable and unstable chromatin structures: the effect of nucleosomal spacing

Coarse-grained models are used to assess the packing of the 30-nm chromatin fiber. First, rigid assembly models for nucleosomal repeats from 155 to 211 bp are built using the crystal structure of the mononucleosome and attached straight stretches of B-DNA. The resulting fiber conformations are analyzed for static clashes and classified into stable and unstable structures. The effect of flexibility and thermal fluctuations is then taken into account by conducting Monte Carlo simulations of chromatin fiber models. Here the DNA is approximated by a flexible polymer chain with Debye–Hückel electrostatics, the geometry of the linker DNA connecting the nucleosomes is based on a two-angle zigzag model, and nucleosomes are represented by flat ellipsoids interacting via an attractive Gay–Berne potential. Unstable fibers occur at a particular repeat length period of 10 bp. Also, the regions of densely compacted fibers repeat at intervals of 10 bp. Besides one- and two-start helical zigzag structures, we show evidence for possible three-start structures, which have not been reported in experiments yet. Finally, we show that a local opening of the linker DNA at the nucleosome core—as probably occurs upon histone acetylation—leads to more open and flexible structures.     

DNA damage in nucleosomes

Eukaryotic genomic DNA is packed into chromatin, whose fundamental structural unit is the nucleosome. As DNA-histone protein complexes, nucleosomes show different properties toward exogenous and endogenous DNA-damaging agents. This review summarizes nucleosome DNA damage due to different sources, including alkylating agents, radicals, UV radiation and reactive DNA damage intermediates. In most cases, the histone core protects the associated DNA against damage via its structure and/or scavenging of damaging agents. In contrast, histones react with damaged DNA and, in some instances, catalyze DNA damage in the nucleosome. The biological consequence of nucleosome DNA damage and future prospects in this field are briefly discussed.     

A 1,2-d(GpG) cisplatin intrastrand cross-link influences the rotational and translational setting of DNA in nucleosomes

The mechanism of action of platinum-based anticancer drugs such as cis-diamminedichloroplatinum(II), or cisplatin, involves three early steps: cell entry, drug activation, and target binding. A major target in the cell, responsible for the anticancer activity, is nuclear DNA, which is packaged in nucleosomes that comprise chromatin. It is important to understand the nature of platinum-DNA interactions at the level of the nucleosome. The cis-{Pt(NH3)2}2+ 1,2-d(GpG) intrastrand cross-link is the DNA lesion most commonly encountered following cisplatin treatment. We therefore assembled two site-specifically platinated nucleosomes using synthetic DNA containing defined cis-{Pt(NH3)2}2+ 1,2-d(GpG) cross-links and core histones from HeLa-S3 cancer cells. The structures of these complexes were investigated by hydroxyl radical footprinting and exonuclease III mapping. Our experiments demonstrate that the 1,2-d(GpG) cross-link alters the rotational setting of the DNA on the histone octamer core such that the lesion faces inward, with disposition angles of the major groove relative to the core of xi approximately -20 degrees and xi approximately 40 degrees . We observe increased solvent accessibility of the platinated DNA strand, which may be caused by a structural perturbation in proximity of the 1,2-d(GpG) cisplatin lesion. The effect of the 1,2-d(GpG) cisplatin adduct on the translational setting of the nucleosomal DNA depends strongly on the position of the adduct within the sequence. If the cross-link is located at a site that is in phase with the preferred rotational setting of the unplatinated nucleosomal DNA, the effect on the translational position is negligible. Minor exonuclease III digestion products in this substrate indicate that the cisplatin adduct permits only those translational settings that differ from one another by integral numbers of DNA helical turns. If the lesion is located out of phase with the preferred rotational setting, the translational position of the main conformation was shifted by 5 bp. Additionally, a fraction of platinated nucleosomes with widely distributed translational positions was observed, suggesting increased nucleosome sliding relative to platinated nucleosomes containing the 1,3-intrastrand d(GpTpG) cross-link investigated previously (Ober, M.; Lippard, S. J. J. Am. Chem. Soc. 2007, 129, 6278-6286).     

Determining the electrophoretic mobility and translational diffusion coefficients of DNA molecules in free solution

The free solution mobility of DNA molecules of different molecular weights, the sequence dependence of the mobility, and the diffusion coefficients of small single- and double-stranded DNA (ss- and dsDNA) molecules can be measured accurately by capillary zone electrophoresis, using coated capillaries to minimize the electroosmotic flow (EOF) of the solvent. Very small differences in mobility between various analytes can be quantified if a mobility marker is used to correct for small differences in EOF between successive experiments. Using mobility markers, the molecular weight at which the free solution mobility of dsDNA becomes independent of molecular weight is found to be approximately 170 bp in 40 mM Tris-acetate-EDTA buffer. A DNA fragment containing 170 bp has a contour length of approximately 58 nm, close to the persistence length of DNA under these buffer conditions. Hence, the approach of the free solution mobility of DNA to a plateau value may be associated with the transition from a rod-like to a coil-like conformation in solution. Markers have also been used to determine that the free solution mobilities of ss- and dsDNA oligomers are sequence-dependent. Double-stranded 20-bp oligomers containing runs of three or more adenine residues in a row (A-tracts) migrate somewhat more slowly than 20-mers without A-tracts, suggesting that somewhat larger numbers of counterions are condensed in the ion atmospheres of A-tract DNAs, decreasing their net effective charge. Single-stranded 20-mers with symmetric sequences migrate approximately 1% faster than their double-stranded counterparts, and faster than single-stranded 20-mers containing A(5)- or T(5)-tracts. Interestingly, the average mobility of two complementary single-stranded 20-mers is equal to the mobility of the double-stranded oligomer formed upon annealing. Finally, the stopped migration method has been used to measure the diffusion coefficients of single- and double-stranded oligomers. The diffusion coefficients of ssDNA oligomers containing 20 nucleotides are approximately 50% larger than those of double-stranded DNA oligomers of the same size, reflecting the greater flexibility of ssDNA molecules. The methods used to carry out these experiments are also described in detail.     

Structure and Dynamics in the Nucleosome Revealed by Solid‐State NMR

Eukaryotic chromatin structure and dynamics play key roles in genomic regulation. In the current study, the secondary structure and intramolecular dynamics of human histone H4 (hH4) in the nucleosome core particle (NCP) and in a nucleosome array are determined by solid‐state NMR (SSNMR). Secondary structure elements are successfully localized in the hH4 in the NCP precipitated with Mg²⁺. In particular, dynamics on nanosecond to microsecond and microsecond to millisecond timescales are elucidated, revealing diverse internal motions in the hH4 protein. Relatively higher flexibility is observed for residues participating in the regulation of chromatin mobility and DNA accessibility. Furthermore, our study reveals that hH4 in the nucleosome array adopts the same structure and show similar internal dynamics as that in the NCP assembly while exhibiting relatively restricted motions in several regions consisting of residues in the N‐terminus, Loop 1, and the α3 helix region.     

On-channel base stacking in microchip capillary gel electrophoresis for high-sensitivity DNA fragment analysis

We evaluated a novel strategy for high-sensitivity DNA fragment analysis in a conventional glass double-T microfluidic chip. The microchip allows for a DNA on-channel concentration based on base stacking (BS) with a microchip capillary gel electrophoretic (MCGE) separation step in a poly(vinylpyrrolidone) (PVP) sieving matrix. Depending if low conductivity caused a neutralization reaction between the hydroxide ions and the run buffer component Tris+, the stacking of DNA fragments were processed in the microchip. Compared to a conventional MCGE separation with a normal electrokinetic injection, the peak heights of 50-2650-base pair (bp) DNA fragments on the MCGE-BS separation were increased 3.9-8.0-fold. When we applied the MCGE-BS method to the analysis of a clinical sample of bovine theileria after PCR reaction, the peak height intensity of the amplified 816-bp DNA fragment from the 18S rRNA of T. buffeli was enhanced 7.0-fold compared to that of the normal injection method.     

Direct Observation of Dynamic Interactions between Orientation-Controlled Nucleosomes in a DNA Origami Frame

The nucleosome is one of the most fundamental units involved in gene expression and consequent cell development, differentiation, and expression of cell functions. We report here a method to place reconstituted nucleosomes into a DNA origami frame for direct observation using high-speed atomic-force microscopy (HS-AFM). By using this method, multiple nucleosomes can be incorporated into a DNA origami frame and real-time movement of nucleosomes can be visualized. The arrangement and conformation of nucleosomes and the distance between two nucleosomes can be designed and controlled. In addition, four nucleosomes can be placed in a DNA frame. Multiple nucleosomes were well accessible in each conformation. Dynamic movement of the individual nucleosomes were precisely monitored in the DNA frame, and their assembly and interaction were directly observed. Neither mica surface modification nor chemical fixation of nucleosomes is used in this method, meaning that the DNA frame not only holds nucleosomes, but also retains their natural state. This method offers a promising platform for investigating nucleosome interactions and for studying chromatin structure.     

Effect of organic cosolvents on the free solution mobility of curved and normal DNA molecules

The free solution mobilities of curved and normal 199-bp DNA fragments have been measured in buffer solutions containing various quantities of the organic cosolvents methanol, ethanol, 2-propanol, 2-methyl-2,4-pentanediol (MPD), ethylene glycol, and ACN, using CE. The curved fragment, taken from the VP1 gene of SV40, contains five unevenly spaced A- and T-tracts in a centrally located "curvature module"; the A- and T-tracts have been mutated to other sequences in the normal 199-bp fragment. The free solution mobility of the curved 199-bp fragment is significantly lower than that of its normal counterpart in aqueous solutions [Stellwagen, E., Lu, Y. J., Stellwagen, N. C., Nucleic Acids Res. 2005, 33, 4425-4432]. The mobilities of both the curved and normal fragments decrease with increasing cosolvent concentration, due to the effect of the cosolvent on the viscosity and dielectric constant of the solution. The mobility differences between the curved and normal 199-bp fragments and the mobility ratios decrease approximately linearly with the increasing mole fraction of cosolvent in the solution. Hence, MPD and other organic cosolvents affect DNA electrophoretic mobility by a common mechanism, most likely the preferential hydration of the DNA surface that occurs in aqueous cosolvents. The gradual loss of the anomalously slow mobility of the curved 199-bp fragment with increasing cosolvent concentration, combined with other data in the literature, suggests that preferential hydration gradually widens the narrow A-tract minor groove, releasing site-bound counterions in the minor groove and shifting the conformation toward that of normal DNA.     

DNA liquid crystalline blue phases. Electron microscopy evidence and biological implications

The isotropic-liquid crystalline transition of concentrated DNA solutions is investigated using freeze-fracture electron microscopy in order to understand the first steps of the DNA condensation process. Between the isotropic liquid and the cholesteric mesophase, we report the existence of double twist DNA bundles and describe their long range ordering into 3D networks. This organization corresponds to the formation of 'blue phases' already observed in thermotropic liquid crystals, but never reported in lyotropic systems. In addition, the size of the DNA molecule, about ten times that of most thermotropic materials, allows here the molecular resolution imaging of blue phase structures. Since such structures recall chromatin organization of some Procaryotes and lower Eucaryotes, we suspect that they may be widespread and of potential interest in the regulation of chromatin functions.     

Dissociation of DNA from histone by reaction of anti-cancer drug cis-diamminedichloroplatinum(II) with DNA-histone complexes used as cellular model

Although both cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) and trans-diamminedichloroplatinum(II) bind to DNA, only cis-DDP is widely used as a chemotherapeutic agent; the stereoisomer trans-DDP is inactive. DNA, generally, is wound around the histone core in the nucleus of living cells and forms the nucleosome structure. To understand the essentially different anticancer activities of cis-DDP and trans-DDP, it is necessary to investigate the interaction of cis-DDP (or trans-DDP) with DNA around the histone in the nucleosome. Here, we used psiX174DNA-histone(LNCaP) complexes prepared by the reaction of psiX174DNA with histone(LNCaP) extracted from LNCaP cells. We first show that the ability of cis-DDP to dissociate the DNA from psiX174DNA-histone(LNCaP), as a nucleosome model, is much stronger than that of trans-DDP. As a result of the action of cis-DDP, the DNA in the nucleosome is rendered naked, and the naked DNA is vulnerable to cis-DDP (or other drugs). This study describes a new model showing that the difference in the activities of cis-DDP and trans-DDP is related to the difference in their abilities to dissociate the DNA from the nucleosome.     

Expression of chirality in columnar hexagonal phases or DNA and nucleosomes

It is surprising to see how eukaryotic chromosomes or sperm nuclei are highly condensed chromatin materials and how they can sometimes present spectacular helical morphologies. We may suspect that these helical shapes originate from the chiral properties of DNA and other components of chromatin. Dense solutions of DNA and nucleosomes can be prepared in vitro to reproduce some of the characteristics of chromatin. They form multiple ordered phases, either mesophases or 3D crystals, that can be useful to analyze precisely how chiral structures can emerge, or not, from interactions between these constitutive elements. We address the question of the competition between twist and hexagonal packing in dense states of DNA, nucleosomes, chromatin and chromosomes. From the microscopic analysis of many examples, we show how the twist arising from the chirality of the objects can be diluted in the phase and/or expelled along twist walls. These walls are either parallel or normal to the direction of the columns. In the first case, we determine that the twist axis lies parallel to one θ₂ direction of the hexagonal network. Helical shapes of chromosomes and bundles of DNA and chromatin may also be consequences of this competition, as illustrated here.     

Estimation of polyacrylamide gel pore size from Ferguson plots of normal and anomalously migrating DNA fragments. I. Gels containing 3% N,N'-methylenebisacrylamide

The mobilities of normal and anomalously migrating DNA fragments were determined in polyacrylamide gels of different acrylamide concentrations, polymerized with 3% N,N'-methylenebisacrylamide as the crosslinker. The DNA samples were a commercially available 123-bp ladder and two molecular weight ladders containing multiple copies of two 147-base pair (bp) restriction fragments, obtained from the MspI digestion of plasmid pBR322. One of the 147 bp fragments is known to migrate anomalously slowly in polyacrylamide gels. Ferguson plots were constructed for all multimer ladders, using both absolute mobilities and relative mobilities with respect to the smallest DNA molecule in each data set. If the retardation coefficients were calculated from the relative mobilities, and the rms radius of gyration was used as the measure of DNA size, the Ogston equations were obeyed and the gel fiber parameters could be calculated. The effective pore sizes of the gels were estimated from the gel concentration at which the mobility of a given DNA molecule was reduced to one-half its mobility at zero gel concentration. The estimated pore radii ranged from approximately 130 nm for 3.5% gels to approximately 70 nm for 10.5% gels. These values are much larger than the pore sizes previously determined for the polyacrylamide matrix.     

Complexation of DNA with cationic surfactants as studied by small-angle X-ray scattering

The phase behaviors of the complex formed by didodecyldimethylammonium bromide(DDAB)and cetyltrimethylammonium bromide(CTAB)interacting with three different types of DNAs,salmon testes DNA(~2000 bp),21-bp double-stranded oligonucleotides(oligo-ds DNA),and 21-nt single-stranded oligonucleotides(oligo-ss DNA)were studied by synchrotron small-angle X-ray scattering.It was found that the DNA length and flexibility,together with the positive/negative charge ratio,determined the final structure.At higher charge ratios,the DNA length exhibited negligible effect.Both oligo-ds DNA and salmon DNA formed inverted hexagonal packing of cylinders with CTAB,as well as bilayered lamella with DDAB.However,at lower charge ratios,oligo-ds DNA formed a distorted hexagonal phase with CTAB and a new structure with DDAB,which was different from the behaviors of salmon DNA.The flexible oligo-ss DNA formed rich structures that were subject to environmental disturbance.Kinetic study also indicated that the structures of the complex formed by oligo-ss DNA took much longer to mature than the structures formed by oligo-ds DNA.We attributed this result to the conformational adjustment of oligo-ss DNA in the complex.     

Platinum drug adduct formation in the nucleosome core alters nucleosome mobility but not positioning

Nucleosome positioning and reorganization regulate DNA site exposure in chromatin. Platinum anticancer agents form DNA adducts that disrupt nuclear activities, triggering apoptosis. Mechanistic insight would aid in the development of improved therapies to circumvent drug toxicity and resistance. We show that platinum adducts formed by reaction of cisplatin or oxaliplatin with the nucleosome core inhibit histone octamer-DNA sliding but do not cause significant alteration of positioning. Thus, adduct formation reinforces positional preferences intrinsic to the DNA sequence, which indicates that modulation of platinum drug site selectivity by histone octamer association may relate to nucleosome-specific properties of DNA. This sheds light on platinum drug-mediated inhibition of chromatin remodeling in vivo and suggests that adducts can shield their own repair and interfere with genomic activities by directly altering nucleosome dynamics.     

Targeted Histone Peptides: Insights into the Spatial Regulation of the Methyltransferase PRC2 by using a Surrogate of Heterotypic Chromatin

Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native chromatin, each nucleosome can differ from its neighbors as a result of covalent modifications to both the DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation. A chemical approach to the production of heterotypic chromatin that can be used in such studies is introduced. This method involves the attachment of a user‐defined modified histone peptide to a designated nucleosome within the polymer by using a peptide nucleic acid (PNA) targeting compound. This strategy was applied to dissect the effect of chromatin context on the activity of the histone methyltransferase PRC2. The results show that PRC2 can be stimulated to produce histone H3 methylation from a defined nucleation site.     

Detection of DNA curvature by transverse pore gradient gel electrophoresis on PhastSystem gels.

It has been known since 1990 that DNA curvature can be recognized on transverse pore gradient gels by an intersection of "Ferguson curves" with those of DNA size standards. The miniaturized PhastSystem polyacrylamide gels allow one to detect DNA curvature effortlessly and fast and at great economy of sample relative to alternative methods of electrophoresis. Using the transverse gradient gel electrophoresis method, it was found that the 660 bp length subfragment of the matrix attachment region (MAR) sequence of the chicken lysosyme gene migrates as a fragment of 800-900 bp length. When subjected to digestion with the restriction enzyme HaeIII, the fragment gives rise to two species of 248 and 412 bp length, respectively. The Ferguson curves of both species intersect with those of DNA size standards, indicating that both exhibit curvature. Only the curvature of the 412 bp fragment conforms to prediction. Ethidium bromide abolishes the effect of curvature on the fragment, reducing its apparent size from 900 to 660, the value obtained by agarose gel electrophoresis.     

DNA cholesteric phases: the role of DNA molecular chirality and DNA-DNA electrostatic interactions

DNA molecules form dense liquid-crystalline twisted phases both in vivo and in vitro. How the microscopic DNA chirality is transferred into intermolecular twist in these mesophases and what is the role of chiral DNA-DNA electrostatic interactions is still not completely clear. In this paper, we first give an extended overview of experimental observations on DNA cholesteric phases and discuss the factors affecting their stability. Then, we consider the effects of steric and electrostatic interactions of grooved helical molecules on the sign of cholesteric twist. We present some theoretical results on the strength of DNA-DNA chiral electrostatic interactions, on DNA-DNA azimuthal correlations in cholesteric phases, on the value of DNA cholesteric pitch, and on the regions of existence of DNA chiral phases stabilized by electrostatic interactions. We suggest for instance that 146 bp long DNA fragments with stronger affinities for the nucleosome formation can form less chiral cholesteric phases, with a larger left-handed cholesteric pitch. Also, the value of left-handed pitch formed in assemblies of homologous DNA fragments is predicted to be smaller than that of randomly sequenced DNAs. We expect also the cholesteric assemblies of several-kbp-long DNAs to require higher external osmotic pressures for their stability than twisted phases of short nucleosomal DNA fragments at the same DNA lattice density.