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.     

Nucleosome Assembly Alters the Accessibility of the Antitumor Agent Duocarmycin B2 to Duplex DNA

To evaluate the reactivity of antitumor agents in a nucleosome architecture, we conducted in vitro studies to assess the alkylation level of duocarmycin B₂ on nucleosomes with core and linker DNA using sequencing gel electrophoresis. Our results suggested that the alkylating efficiencies of duocarmycin B₂ were significantly decreased in core DNA and increased at the histone‐free linker DNA sites when compared with naked DNA conditions. Our finding that nucleosome assembly alters the accessibility of duocarmycin B₂ to duplex DNA could advance its design as an antitumor agent.     

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.     

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.     

Damage control of DNA in nucleosome core particles: when a histone's loving, protective embrace is just not good enough

Packaging DNA into nucleosome core particles generally offers protection from damage by molecules diffusing in solution. However, on page 403 of this issue, Barton and coworkers report that although noncovalently bound, activated Rh (Rhodium) does not readily bind within nucleosomal DNA, activated Rh that is covalently tethered to the 5' terminus of a histone-associated oligonucleotide oxidizes guanine bases from a distance of up to 24 base pairs, demonstrating that histones do not protect DNA from long-range damage from the transport of charge through stacked bases. This implies that oxidative damage generated on DNA in vivo may spread from an initially damaged site to distal sites. Once created, such sites may persist and be resistant to repair because of the protective packaging by histones; they thus may result in permanent mutations.     

Damage to rat retinal DNA induced in vivo by visible light

Intense visible light can damage retinal photoreceptor cells by photochemical or thermal processes, leading to cell death. The precise mechanism of light-induced damage is unknown; however, oxidative stress is thought to be involved, based on the protective effect of antioxidants on the light-exposed retina. To explore the in vivo effects of light on retinal DNA, rats were exposed to intense visible light for up to 24 h and the time courses of single-strand breaks in restriction fragments containing the opsin, insulin 1 and interleukin-6 genes were measured. All three gene fragments displayed increasing single-strand modifications with increasing light exposure. Treatment with the antioxidant dimethylthiourea prior to light exposure delayed the development of net damage. The time course of double-strand DNA damage was also examined in specific genes and in repetitive DNA. The appearance of discrete 140-200 base-pair DNA fragments after 20 h of light exposure implicated a nonrandom, possibly enzymatic damaging mechanism. The generation of nucleosome core-sized DNA fragments, in conjunction with single-strand breaks, suggests two phases of light-induced retinal damage, with random attack on DNA by activated oxygen species preceding enzymatic degradation.     

Evaluation of the protective capabilities of nucleosome STRs obtained by large‐scale sequencing

Partial DNA profiles are often obtained from degraded forensic samples and are hard to analyze and interpret. With in‐depth studies on degraded DNA, an increasing number of forensic scientists have focused on the intrinsic structural properties of DNA. In theory, nucleosomes offer protection to the bound DNA by limiting access to enzymes. In our study, we performed large‐scale DNA sequencing on nucleosome core DNA of human leucocytes. Five nucleosome short tandem repeats (STRs) were selected (including three forensic common STRs (i.e. TPOX, TH01, and D10S1248) and two unpublished STRs (i.e. AC012568.7 and AC007160.3)). We performed a population genetic investigation and forensic genetic statistical analysis of these two unpublished loci on 108 healthy unrelated individuals of the HeBei Han population in China. We estimated the protective capabilities of five selected nucleosome loci and MiniFiler? loci with artificial degraded DNA and case samples. We also analyzed differences between sequencing results and software predicted results. Our findings showed that nucleosome STRs were more likely to be detected than MiniFiler? loci. They were well protected from degradation by nucleosomes and could be candidates for further nucleosome multiplex construction, which would increase the chances of obtaining a better balanced profile with fewer allelic drop‐outs.     

Nucleosomes and cisplatin

In order to form a covalent complex with DNA inside human cells, cisplatin has to overcome the protective environment of a nucleosome, where DNA is complexed with histone proteins. Todd and Lippard (2010) expand our understanding of this process by describing the structure of a nucleosome containing a Pt-DNA adduct, which has important implications for more effective chemotherapeutic drug development.     

THE EFFECTS OF ULTRAVIOLET C ON in vitro NUCLEOSOME ASSEMBLY AND STABILITY

Abstract The effects of ultraviolet C (UVC) irradiation on nucleosome assembly and its stability were investigated quantitatively using an in vitro nucleosome assembly system comprising a plasmid DNA of pBR322 and core histones isolated from rat ascites hepatoma cells. Nucleosomal formation was estimated by analyzing the resulting DNA supercoils. When UVC-irradiated (3000 J/m²) DNA was used as a substrate for the nucleosome assembly system, the nucleosomal formation efficiency was reduced by half compared with nonirradiated DNA. On the other hand, when the reconstituted nucleosomes (minichromosomes) on the nonirradiated DNA were irradiated with UVC (3000 J/m²), about half each were disrupted and retained. These results indicate that it is difficult for UV-damaged DNA regions to supercoil around the histone octamers to form nucleosomes and that the histone octamers in the UV-damaged nucleosomes tend to be dissociated from DNA.     

UV INDUCED (6-4) PHOTOPRODUCTS ARE DISTRIBUTED DIFFERENTLY THAN CYCLOBUTANE DIMERS IN NUCLEOSOMES

We have compared the distributions of two stable UV photoproducts in nucleosome core DNA at the single-nucleotide level using a T4 polymerase-exonuclease mapping procedure. The distribution of pyrimidine-pyrimidone (6-4) dimers was uncovered by reversing the major UV photo-product, cis-syn cyclobutane pyrimidine dimer, with E. coli DNA photolyase and photoreactivating light. Whereas the distribution of total UV photoproducts in nucleosome core DNA forms a striking 10.3 base periodic pattern, the distribution of (6-4) dimers is much more random throughout the nucleosome core domain. Therefore, histone-DNA interactions in nucleosomes strongly modulate formation of the major class of UV-induced photoproducts, while having either a constant effect or no effect on (6-4) dimer formation.     

Cisplatin damage overrides the predefined rotational setting of positioned nucleosomes

Cisplatin and carboplatin are used successfully to treat various types of cancer. The drugs target the nucleosomes of cancer cells and form intrastrand DNA cross-links that are located in the major groove. We constructed two site-specifically modified nucleosomes containing defined intrastrand cis-{Pt(NH3)2}(2+) 1,3-d(GpTpG) cross-links. Histones from HeLa-S3 cancer cells were transferred onto synthetic DNA duplexes having nucleosome positioning sequences. The structures of these complexes were investigated by hydroxyl radical footprinting. Employing nucleosome positioning sequences allowed us to quantify the structural deviation induced by the cisplatin adduct. Our experiments demonstrate that a platinum cross-link locally overrides the rotational setting predefined in the nucleosome positioning sequence such that the lesion faces toward the histone core. Identical results were obtained for two DNA duplexes in which the sites of platination differed by approximately half a helical turn. Additionally, we determined that cisplatin unwinds nucleosomal DNA globally by approximately 24 degree. The intrastrand cis-{Pt(NH3)2}(2+) 1,3-d(GpTpG) cross-links are located in an area of the nucleosome that contains locally overwound DNA in undamaged reference nucleosomes. Because most nucleosome positions in vivo are defined by the intrinsic DNA sequence, the ability of cisplatin to influence the structure of these positioned nucleosomes may be of physiological relevance.     

Site-specific recognition of guanosine by manganese(III) corroles in DNA non-duplex regions through active oxygen transfer

DNA damage plays an important role in cellular processes. Besides natural protein nucleases, different types of efficient agents for DNA damage have been developed over recent decades in the search for new anticancer and antiviral drugs. In addition to the double-stranded configuration, DNA structures also include some non-duplex regions, which are considered to be from spontaneous errors in DNA replication, thus playing an important role for cells. Herein, we focused on these non-duplex regions of DNA and generated manganese(III) corroles, which exhibit a highly selective cleavage ability for guanosine units located at non-duplex portions, such as loops and bulges. The cleavage mechanism was demonstrated to be a manganese-induced oxidation process. The results given herein show a molecular approach that could specifically probe the guanosine units in DNA non-duplex structures, thus representing a promising step in the construction of tools to target non-duplex structures in chromosomes.     

Chromatin and Nucleosome Organizations and DNA Replication of Nucleus Reassembled in vitro Using Purified Exogenous DNA and Xenopus Egg Extracts

It has been demonstrated in the last ten years that the nuclear reassembly may occur in the cell-free systems from frog egg extracts added with exogenous naked DNA. However, there remains an open question : is the cell-free reassembled nucleus structurally similar to the nucleus in the intact cell ? That is, does the cell-free reassembled nucleus contain nucleosomes and chromatin? For this issue, we have designed experiments for identifying the internal structures of the cell-free reassembled nucleus. These experiments show that the nucleus reassembled in vitro also contains chromatin which is composed of typical 10 nm nucleosome fibers of "beads-on-a-string", 30 nm filaments and the next higher-order structures. The digestion experiment with the enzyme micrococcal nuclease has demonstrated that the DNA in the nucleosome of the reconstituted chromatin is about 200 base pairs (bp) in length, of which 165 bp may be in the nucleosome particle, and 35 bp may be in the linker between two particles. Prolongin     

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.     

Nucleosome Immobilization Strategies for Single‐Pair FRET Microscopy

All genomic transactions in eukaryotes take place in the context of the nucleosome, the basic unit of chromatin, which is responsible for DNA compaction. Overcoming the steric hindrance that nucleosomes present for DNA‐processing enzymes requires significant conformational changes. The dynamics of these have been hard to resolve. Single‐pair Fluorescence Resonance Energy Transfer (spFRET) microscopy is a powerful technique for observing conformational dynamics of the nucleosome. Nucleosome immobilization allows the extension of observation times to a limit set only by photobleaching, and thus opens the possibility of studying processes occurring on timescales ranging from milliseconds to minutes. It is crucial however, that immobilization itself does not introduce artifacts in the dynamics. Here we report on various nucleosome immobilization strategies, such as single‐point attachment to polyethylene glycol (PEG) or surfaces coated with bovine serum albumin (BSA), and confinement in porous agarose or polyacrylamide gels. We compare the immobilization specificity and structural integrity of immobilized nucleosomes. A crosslinked star polyethylene glycol coating performs best with respect to tethering specificity and nucleosome integrity, and enables us to reproduce for the first time bulk nucleosome unwrapping kinetics in single nucleosomes without immobilization artifacts.     

Structural variability of nucleosomes detected by single-pair Förster resonance energy transfer: histone acetylation, sequence variation, and salt effects

Nucleosomes were reconstituted from 170 bp long fragments of 5S rDNA and an optimal positioning sequence, the Selex 601, with recombinant histones. In free-solution single pair F?rster resonance energy transfer (spFRET) measurements of the distance between fluorescently labeled bases in the nucleosomal DNA, the samples exhibited structural diversity. The structural heterogeneity correlated with the stability of the complexes and depended on the DNA sequence and histone acetylation. The stability of the nucleosomes was assessed via dilution-driven disruption: histone acetylation decreased nucleosome stability. The spFRET experiments used a new approach for data acquisition and analysis that we term "deliberately detuned detection" (D3). This permits the separation of subpopulations in the samples even for the low-FRET regime characteristic for the linker-DNA labeled nucleosomes. Thus, it became possible to study in more detail histone acetylation- and salt-dependent structural variations using either end- or internally labeled DNAs on the nucleosome. We found that the distance distribution of the fluorophore pairs on the linker DNA ends was much more sensitive to histone acetylation or sequence variation than that of labels on the internal part of the DNA, which was more tightly associated with the histone core. spFRET on freely diffusing nucleosomes allows us therefore to localize the influence of histone modifications and DNA sequence variations on the nucleosome structure and dynamics.     

DNA packing in chromatine, a manifestation of the Bonnet transformation

The packing of DNA is described using the formalism of differential geometry. Winding of the DNA double helix around the histone 2-5 octamer forming a nucleosome and the condensation of the so-formed bead-on-a-string chromatine aided by histone 1 is interpreted as two consecutive isometric, i.e. Bonnet, transformations. The DNA double helix can be approximated to a helicoid which can be transformed isometrically to a catenoid, an approximation of the nucleosome. Owing to the organization of the histone octamer the extended chromatine takes a helicoidal shape allowing a second Bonnet transformation to consummate the condensation into a chromatine fibre.     

DNA interstrand cross-link formation initiated by reaction between singlet oxygen and a modified nucleotide

DNA is the target of many anti-cancer therapies. These agents damage the biopolymer by oxidation or by alkylation. Interstrand DNA cross-links are believed to be the source of cytotoxicity of anti-tumor agents, such as mitomycin C, which alkylate the biopolymer. In contrast, deoxyguanosine oxidation is the result of reaction between DNA and singlet oxygen, which is the damaging species produced in photodynamic therapy. We have shown that, upon oxidation by singlet oxygen, an analogue of thymidine (2) rearranges to a methide, which forms DNA-DNA interstrand cross-links. This novel process suggests that 2 may be a useful adjuvant in photodynamic therapy.     

Preferential 5‐Methylcytosine Oxidation in the Linker Region of Reconstituted Positioned Nucleosomes by Tet1 Protein

Tet (ten–eleven translocation) family proteins oxidize 5‐methylcytosine (mC) to 5‐hydroxymethylcytosine (hmC), 5‐formylcytosine (fC), and 5‐carboxycytosine (caC), and are suggested to be involved in the active DNA demethylation pathway. In this study, we reconstituted positioned mononucleosomes using CpG‐methylated 382 bp DNA containing the Widom 601 sequence and recombinant histone octamer, and subjected the nucleosome to treatment with Tet1 protein. The sites of oxidized methylcytosine were identified by bisulfite sequencing. We found that, for the oxidation reaction, Tet1 protein prefers mCs located in the linker region of the nucleosome compared with those located in the core region.