Superhelicity of nucleosomal DNA changes its double-helical repeat

Winding DNA in a superhelix can be considered a process consisting of two smooth deformations: bending and twisting. The extra twist angle introduced by winding DNA into the nucleosomal superhelix is calculated by means of the Crick formula to be -0.5 degrees per base pair (bp). This is equivalent to a change of -0.15 +/- 0.015 bp in the DNA double-helical repeat. Free DNA in solution is known to have a helical repeat of 10.55 +/- 0.1 bp. On the other hand, a weighted average of various estimates of the DNA repeat in the nucleosome is 10.38 +/- 0.02. The difference happens to be perfectly accounted for by the superhelicity of the nucleosomal DNA. This implies that the latter is essentially nonconstrained .     

A defined structure of the 30 nm chromatin fibre which accommodates different nucleosomal repeat lengths

Earlier work on the condensation of chromatins of different repeat lengths into the 30 nm fibre has been surveyed and it is shown that the external geometry of the fibre must be the same for all the chromatins. This can only be fitted by a helical coiling of nucleosomes into a solenoid with the linker DNA disposed internally. On this basis, various models were calculated and compared with published electric dichroism data. The only good fit is found with a 'reverse-loop' model, where the linker DNA forms a complete turn into the hole of the solenoid, of opposite hand to the nucleosomal DNA superhelix. This gives a topological linking number of one per nucleosome and would resolve the 'linking number paradox' if the DNA screw is the same in chromatin as in solution. The feasibility of a reverse-loop for short linkers (down to 15 base pairs) was investigated by model building and kinks of approximately 120 degrees into both DNA grooves are described, which will allow such packing. There will, however, be a 'forbidden' range for the linker DNA length, between approximately 1 and 14 bp, corresponding to nucleosomal repeats of 163 and 176 bp.     

Molecular Dynamics Simulations of a Nucleosome and Free DNA

Abstract

All atom molecular dynamics simulations (10ns) of a nucleosome and of its 146 basepairs of DNA free in solution have been conducted. DNA helical parameters (Roll, Tilt, Twist, Shift, Slide, Rise) were extracted from each trajectory to compare the conformation, effective force constants, persistence length measures, and fluctuations of nucleosomal DNA to free DNA. The conformation of DNA in the nucleosome, as determined by helical parameters, is found to be largely within the range of thermally accessible values obtained for free DNA. DNA is found to be less flexible on the nucleosome than when free in solution, however such measures are length scale dependent. A method for disassembling and reconstructing the conformation and dynamics of the nucleosome using Fourier analysis is presented. Long length variations in the conformation of nucleosomal DNA are identified other than those associated with helix repeat. These variations are required to create a proposed tetrasome conformation or to qualitatively reconstruct the 1.75 turns of the nucleosome's superhelix. Reconstruction of free DNA using selected long wavelength variations in conformation can produce either a left-handed or a right-handed superhelix. The long wavelength variations suggest 146 basepairs is a natural length of DNA to wrap around the histone core.     

Structure-based Analysis of DNA Sequence Patterns Guiding Nucleosome Positioning in vitro

Abstract

Recent studies of genome-wide nucleosomal organization suggest that the DNA sequence is one of the major determinants of nucleosome positioning. Although the search for underlying patterns encoded in nucleosomal DNA has been going on for about 30 years, our knowledge of these patterns still remains limited. Based on our evaluations of DNA deformation energy, we developed new scoring functions to predict nucleosome positioning. There are three principal differences between our approach and earlier studies: (i) we assume that the length of nucleosomal DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich motifs reflect sequence-dependent interactions between histone arginines and DNA in the minor groove. Using these functions, we analyzed 20 nucleosome positions mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, the pGUB plasmid, chicken β-globin and three 5S rDNA genes). We predicted 15 of the 20 positions with 1-bp precision, and two positions with 2-bp precision. The predicted position of the ‘601’ nucleosome (i.e., the optimum of the computed score) deviates from the experimentally determined unique position by no more than 1 bp—an accuracy exceeding that of earlier predictions.

Our analysis reveals a clear heterogeneity of the nucleosomal sequences which can be divided into two groups based on the positioning ‘rules’ they follow. The sequences of one group are enriched by highly deformable YR/YYRR motifs at the minor-groove bending sites SHL ±3.5 and ±5.5, which is similar to the α-satellite sequence used in most crystallized nucleosomes. Apparently, the positioning of these nucleosomes is determined by the interactions between histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group (that includes the ‘601’ clone) the same YR/YYRR motifs occur predominantly at the sites SHL ±1.5. The interaction between the H3/H4 tetramer and the central part of the nucleosomal DNA is likely to be responsible for the positioning of nucleosomes of this group, and the DNA trajectory in these nucleosomes may differ in detail from the published structures.

Thus, from the stereochemical perspective, the in vitro nucleosomes studied here follow either an X-ray-like pattern (with strong deformations in the terminal parts of nucleosomal DNA), or an alternative pattern (with the deformations occurring predominantly in the central part of the nucleosomal DNA). The results presented here may be useful for genome-wide classification of nucleosomes, linking together structural and thermodynamic characteristics of nucleosomes with the underlying DNA sequence patterns guiding their positions.     

Topography of the ISW2-nucleosome complex: insights into nucleosome spacing and chromatin remodeling

Linker DNA was found to be critical for the specific docking of ISW2 with nucleosomes as shown by mapping the physical contacts of ISW2 with nucleosomes at base-pair resolution. Hydroxyl radical footprinting revealed that ISW2 not only extensively interacts with the linker DNA, but also approaches the nucleosome from the side perpendicular to the axis of the DNA superhelix and contacts two disparate sites on the nucleosomal DNA from opposite sides of the superhelix. The topography of the ISW2-nucleosome was further delineated by finding which of the ISW2 subunits are proximal to specific sites within the linker and nucleosomal DNA regions by site-directed DNA photoaffinity labeling. Although ISW2 was shown to contact approximately 63 bp of linker DNA, a minimum of 20 bp of linker DNA was required for stable binding of ISW2 to nucleosomes. The remaining approximately 43 bp of flanking linker DNA promoted more efficient binding under competitive binding conditions and was functionally important for enhanced sliding of nucleosomes when ISW2 was significantly limiting.     

Assembly of Plasmid DNA into Liposomes After Condensation by Cationic Lipid in Anionic Detergent Solution

A novel strategy to prepare negatively charged and small DNA-containing liposomes after condensation of plasmid DNA by a cationic lipid in deoxycholate micelle environment is described. The average diameter of resulting complexes was 62±8 nm. DNA-containing liposomes were then prepared by dialysis. The shape of the resulting liposomes was spherical. The average diameter and the surface charge of the liposomes were 86±6 nm and −24±3 mV, respectively. The plasmid DNA inside liposomes remained in a supercoiled form after incubation with DNase.     

Molecular and cytogenetic characterization of an AT-rich satellite DNA family in <Emphasis Type="Italic">Urvillea chacoensis</Emphasis> Hunz. (Paullinieae,Sapindaceae)

Urvillea chacoensis is a climber with 2n = 22 and some terminal AT-rich heterochromatin blocks that differentiate it from other species of the genus. The AT-rich highly repeated satellite DNA was isolated from U. chacoensis by the digestion of total nuclear DNA with HindIII and XbaI and cloned in Escherichia coli. Satellite DNA structure and chromosomal distribution were investigated. DNA sequencing revealed that the repeat length of satDNA ranges between 721 and 728 bp, the percentage of AT-base pairs was about 72–73% and the studied clones showed an identity of 92.5–95.9%. Although this monomer has a tetranucleosomal size, direct imperfect repetitions of ~180 bp subdividing it in four nucleosomal subregions were observed. The results obtained with FISH indicate that this monomer usually appears distributed in the terminal regions of most chromosomes and is associated to heterochromatin blocks observed after DAPI staining. These observations are discussed in relation to the satellite DNA evolution and compared with other features observed in several plant groups.     

Alignment of nucleosomes along DNA and organization of spacer DNA in Drosophila chromatin

A series of mono- and dinucleosomal DNAs characterized by an about ten-base periodicity in the size were revealed in the micrococcal nuclease digests of Drosophila chromatin which have 180 +/- 5 base pair (bp) nucleosomal repeat. 20, 30, and 40 bp spacers were found to be predominant in chromatin by trimming DNA in dinucleosomes to the core position. Among several identified mononucleosomes (MN), MN170, MN180 and MN190 were isolated from different sources (the figures indicate the DNA length in bp). The presence of the 10, 20, and 30 bp long spacers was shown in these mononucleosomes by crosslinking experiments. The interaction of histone H3 with the spacer in the Drosophila MN180 particle was also shown by the crosslinking /5/. We conclude from these results that the 10 n bp long intercore DNA (n = 2, 3 and 4) is organized by histone H3, in particular, and together with the core DNA forms a continuous superhelix. Taken together, these data suggest that Drosophila chromatin consists of the regularly aligned and tightly packed MN180, as a repeating unit, containing 10 and 20 bp spacers at the ends of 180 bp DNA. Within the asymmetric and randomly oriented in chromatin MN180, the cores occupy two alternative positions spaced by 10 bp.     

Prenatal appearance of a short nucleosomal DNA repeat length in neurons of the guinea pig cerebral cortex

The organization of chromatin in neurons of the cerebral cortex of the guinea pig brain was analyzed by digesting isolated nuclei with micrococcal nuclease. During development, cortical neurons were observed to undergo an alteration in chromatin structure which results in an atypically short nucleosomal DNA repeat length of 164 bp. This change in chromatin organization occurs postnatally in certain mammals but in the guinea pig it takes place prior to birth between days 32 and 44 of fetal development. This suggests that the appearance of the short nucleosomal DNA repeat length in cortical neurons correlates to a particular stage of differentiation of cortical neurons rather than to the event of birth.     

Molecular dynamics simulations of a nucleosome and free DNA

All atom molecular dynamics simulations (10ns) of a nucleosome and of its 146 basepairs of DNA free in solution have been conducted. DNA helical parameters (Roll, Tilt, Twist, Shift, Slide, Rise) were extracted from each trajectory to compare the conformation, effective force constants, persistence length measures, and fluctuations of nucleosomal DNA to free DNA. The conformation of DNA in the nucleosome, as determined by helical parameters, is found to be largely within the range of thermally accessible values obtained for free DNA. DNA is found to be less flexible on the nucleosome than when free in solution, however such measures are length scale dependent. A method for disassembling and reconstructing the conformation and dynamics of the nucleosome using Fourier analysis is presented. Long length variations in the conformation of nucleosomal DNA are identified other than those associated with helix repeat. These variations are required to create a proposed tetrasome conformation or to qualitatively reconstruct the 1.75 turns of the nucleosome's superhelix. Reconstruction of free DNA using selected long wavelength variations in conformation can produce either a left-handed or a right-handed superhelix. The long wavelength variations suggest 146 basepairs is a natural length of DNA to wrap around the histone core.     

Nucleosome DNA repeat length and histone complement in a fungus exhibiting condensed chromatin

Fungal chromatins are reported to exhibit unusually short nucleosomal DNA repeat lengths. To test whether this is a phylogenetic feature of fungi or rather is correlated with an apparent absence of condensed chromatin in the organisms studied, we have examined the chromatin organization and the complement of basic nuclear proteins in the fungus Entomophthora, an organism which exhibits marked chromatin condensation. Micrococcal nuclease digestion of Entomophthora chromatin revealed a nucleosomal DNA repeat length of 197 +/- 1.2 base pairs (bp). This repeat length is 20-40 bp longer than that reported for any fungus. Entomophthora nucleosomes exhibited an HI-like protein which was much less basic than the HI histones reported for higher eukaryotes but which was similar in basicity to the HI histone reported for the fungus Neurospora. However, the nucleosomal DNA repeat length of Neurospora chromatin is reported to be unusually short, whereas that of Entomophthora was found to be typical of the repeat lengths observed for chromatins of higher eukaryotes. Thus, repeat length, at least in fungi, would not appear to be directly determined by the basicity of the fungal cognate of histone HI.     

AFM imaging and theoretical modeling studies of sequence-dependent nucleosome positioning

Telomeric chromatin has different features with respect to bulk chromatin, since nucleosomal repeat along the chain is unusually short. We studied the role of telomeric DNA sequences on nucleosomal spacing in a model system. Nucleosomal arrays, assembled on a 1500-bp-long human telomeric DNA and on a DNA fragment containing 8 copies of the 601 strong nucleosome positioning sequence, have been studied at the single molecule level, by atomic force microscopy imaging. Random nucleosome positioning was found in the case of human telomeric DNA. On the contrary, nucleosome positioning on 601 DNA is characterized by preferential positions of nucleosome dyad axis each 200 bp. The AFM-derived nucleosome organization is in satisfactory agreement with that predicted by theoretical modeling, based on sequence-dependent DNA curvature and flexibility. The reported results show that DNA sequence has a main role, not only in mononucleosome thermodynamic stability, but also in the organization of nucleosomal arrays.     

An Overview of DNA and RNA Bindings to Antioxidant Flavonoids

In this report we are examining how the antioxidant flavonoids can prevent DNA damage and what mechanism of action is involved in the process. Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. We study the interactions of quercetin (que), kaempferol (kae), and delphinidin (del) with DNA and transfer RNA in aqueous solution at physiological conditions, using constant DNA or RNA concentration 6.25 mmol (phosphate) and various pigment/polynucleotide(phosphate) ratios of 1/65 to 1 (DNA) and 1/48 to 1/8 (tRNA). The structural analysis showed quercetin, kaempferol, and delphinidin intercalate DNA and RNA duplexes with minor external binding to the major or minor groove and the backbone phosphate group with overall binding constants for DNA adducts K _que = 7.25 (±0.65) × 10⁴ M⁻¹, K _kae = 3.60 (±0.33) × 10⁴ M^−1, and K _del = 1.66 (±0.25) × 10⁴ M⁻¹ and for tRNA adducts K _que = 4.80 (±0.50) × 10⁴ M⁻¹, K _kae = 4.65 (±0.45) × 10⁴ M^−1, and K _del = 9.47 (±0.70) × 10⁴ M⁻¹. The stability of adduct formation is in the order of del>que>kae for tRNA and que>kae>del for DNA. Low flavonoid concentration induces helical stabilization, whereas high pigment content causes helix opening. A partial B to A-DNA transition occurs at high drug concentration, while tRNA remains in A-family structure. The antioxidant activity of flavonoids changes in order delphinidin>quercetin>kaempferol. The results show intercalated flavonoids can make them strong antioxidants to protect DNA from harmful free radical reactions.     

Topological analysis of plasmid chromatin from yeast and mammalian cells

Yeast has proven to be a powerful system for investigation of chromatin structure. However, the extent to which yeast chromatin can serve as a model for mammalian chromatin is limited by the significant number of differences that have been reported. To further investigate the structural relationship between the two chromatins, we have performed a DNA topological analysis of pRSSVO, a 5889 base-pair plasmid that can replicate in either yeast or mammalian cells. When grown in mammalian cells, pRSSVO contains an average of 33 negative supercoils, consistent with one nucleosome per 181 bp. This is close to the measured nucleosome repeat length of 190 bp. However, when grown in yeast cells, pRSSVO contains an average of only 23 negative supercoils, which is indicative of only one nucleosome per 256 bp. This is dramatically different from the measured nucleosome repeat length of 165 bp. To account for these observations, we suggest that yeast chromatin is composed of relatively short ordered arrays of nucleosomes with a repeat of 165 bp, separated by substantial gaps, possibly corresponding to regulatory regions.     

Surprising Twists in Nucleosomal DNA with Implication for Higher-order Folding

While nucleosomes are dynamic entities that must undergo structural deformations to perform their functions, the general view from available high-resolution structures is a largely static one. Even though numerous examples of twist defects have been documented, the DNA wrapped around the histone core is generally thought to be overtwisted. Analysis of available high-resolution structures from the Protein Data Bank reveals a heterogeneous distribution of twist along the nucleosomal DNA, with clear patterns that are consistent with the literature, and a significant fraction of structures that are undertwisted. The subtle differences in nucleosomal DNA folding, which extend beyond twist, have implications for nucleosome disassembly and modeled higher-order structures. Simulations of oligonucleosome arrays built with undertwisted models behave very differently from those constructed from overtwisted models, in terms of compaction and inter-nucleosome contacts, introducing configurational changes equivalent to those associated with 2–3 base-pair changes in nucleosome spacing. Differences in the nucleosomal DNA pathway, which underlie the way that DNA enters and exits the nucleosome, give rise to different nucleosome-decorated minicircles and affect the topological mix of configurational states.     

Structure of nucleosomes and organization of internucleosomal DNA in chromatin

We have compared the mononucleosomal pattern produced by micrococcal nuclease digestion of condensed and unfolded chromatin and chromatin in nuclei from various sources with the repeat length varying from 165 to 240 base-pairs (bp). Upon digestion of isolated H1-containing chromatin of every tested type in a low ionic strength solution (unfolded chromatin), a standard series of mononucleosomes (MN) was formed: the core particle, MN145, and H1-containing, MN165, MN175, MN185, MN195, MN205 and MN215 (the indexes give an approximate length of the nucleosomal DNA that differs in these particles by an integral number of 10 bp). In addition to the pattern of unfolded chromatin, digestion of whole nuclei or condensed chromatin (high ionic strength of Ca2+) gave rise to nuclei-specific, H1-lacking MN155. Digestion of H1-lacking chromatin produced only MN145, MN155 and MN165 particles, indicating that the histone octamer can organize up to 165 bp of nucleosomal DNA. Although digestion of isolated sea urchin sperm chromatin (repeat length of about 240 bp) at a low ionic strength gave a typical "unfolded chromatin pattern", digests of spermal nuclei contained primarily MN145, MN155, MN235 and MN245 particles. A linear arrangement of histones along DNA (primary organization) of the core particle was found to be preserved in the mononucleosomes, with the spacer DNA length from 10 to 90 bp on one (in MN155) or both sides of core DNA being a multiple of about 10 bp. In MN235, the core particle occupies preferentially a central position with the length of the spacer DNA on both sides of the core DNA being usually about 30 + 60 or 40 + 50 bp. Histone H1 is localized at the ends of these particles, i.e. close to the centre of the spacer DNA. The finding that globular part of histones H3 and sea urchin sperm H2B can covalently bind to spacer DNA suggests their involvement in the organization of chromatin superstructure. Our data indicate that decondensation of chromatin is accompanied by rearrangement of histone H1 on the spacer DNA sites adjacent to the core particle and thus support a solenoid model for the chromatin superstructure in nuclei in which the core DNA together with the spacer DNA form a continuous superhelix.     

Preliminary characterization of a thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41

A thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41 was characterized in this study. The polI was cloned, sequenced and over-expressed in Escherichia coli. The expressed 110 kDa fusion protein of PolI was stable at 70°C for 1 h. Compared with DNA polymerase I of E. coli (TaKaRa), the relative polymerase activity of this PolI was 3.33 ± 0.1 RFU μl⁻¹ at 37°C using fluorescent quantitative analysis. It showed higher polymerase activity than E. coli PolI at higher temperature, with a relative activity of 3.75 ± 0.1 RFU μl⁻¹ at 70°C. The polI sequence analysis and the protein structure prediction indicated that this protein had a high similarly to other PolI from thermophilic micro-organisms. This information is of importance for future study for evolution of the house-keeping gene polI in entomopathogenic bacterium B. sphaericus.     

Binding of the transition metal ion [(H20)(NH3)5Ru(II)]2+ to nucleosomal core and internucleosomal DNA

The goal of this study is to establish the nature of pentammineruthenium(III) binding to DNA in intact mouse liver nuclei. Also, we wish to determine whether the nucleosomal organization of mouse chromatin has a substantial effect on the relative Ru(III) binding levels of internucleosomal and nucleosomal core DNA. These questions are important because ammineruthenium compounds share chemical and biological properties with the cis-dichlorodiammineplatinum(II) or cisplatin chemotherapeutic agent. Therefore, they represent a potential class of new chemotherapeutic agents. We find that in intact nuclei the predominant DNA binding site for pentammineruthenium(II), followed by air oxidation to pentammineruthenium(III), is N-7 guanine, as is the case with cisplatin. Also, the Ru(III) distribution between internucleosomal and nucleosomal core DNA was found to be nearly identical as probed with three non-specific deoxyribonucleases.