DNA In Nucleosome Core Particles Research Articles

Oxidative Defect Detection Within Free and Packed DNA Systems: A Quantum Mechanical/Molecular Mechanics (QM/ MM) Approach.

Base excision repair enzymes (BERs) detect and repair oxidative DNA damage with efficacy despite the small size of the defects and their often only minor structural impact. A charge transfer (CT) model for rapid scanning of DNA stretches has been evoked to explain the high detection rate in the face of numerous, small lesions. The viability of CT DNA defect detection is explored via hybrid QM/MM computational studies that leverage the accuracy of quantum mechanics (QM) for a region of interest and the descriptive power of molecularmechanics (MM) for the remainder of the system. We find that the presence of an oxidative lesion lowers theredox free energy of oxidation by approximately 1.0 eV regardless of DNA compaction (free DNA versus packed DNA in nucleosome core particles) and damage location indicating the high feasibility of a CT-based process for defect detection in DNA.

DNA conformation and energy in nucleosome core: a theoretical approach

DNA conformation in complex with proteins is far from its canonical B-form. The affinity of complex formation and structure of DNA depend on its attachment configuration and sequence. In this article, we develop a mechanical model to address the problem of DNA structure and energy under deformation. DNA in nucleosome core particle is described as an example. The structure and energy of nucleosomal DNA is calculated based on its sequence and positioning state. The inferred structure has remarkable similarity with X-ray data. Although there is no sequence-specific interaction of bases and the histone core, we found considerable sequence dependency for the nucleosomal DNA positioning. The affinity of nucleosome formation for several sequences is examined and the differences are compatible with observations. We argue that structural energy determines the natural state of nucleosomal DNA and is the main reason for affinity differences in vitro. This theory can be utilized for the DNA structure and energy determination in protein–DNA complexes in general.An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:17

Visualizing DNA Disassociation from the Nucleosome Core Particle

The organization of DNA in nucleosome core particles (NCP) is understood to play crucial regulatory roles in transcription, replication, recombination and repair. High resolution crystal structures deliver detailed snapshots of the NCP in its most stable conformations and reveal thousands of electrostatic interactions that mediate the stability of NCPs. However, many of the techniques that have been applied thus far are ill-suited for directly monitoring the intermediary and dissociated conformations of the NCP. To this end, we applied small angle X-ray scattering (SAXS) - a powerful technique for delivering low resolution structural details of biological molecules. Since SAXS studies of protein-nucleic acid complexes are complicated by the differences in scattering lengths between proteins and nucleic acids, we applied a contrast variation approach where we matched the solvent and protein contrasts to effectively probe the DNA component of the NCP alone. We systematically modulated the electrostatic interactions by adjusting the salt concentration of the solvent and visualized the conformational transition of the DNA from a bound to unbound state during NCP disassembly.

54 DNA conformation and energy in nucleosome core: a theoretical approach

In this work, we developed a mechanical model to address the problem of DNA structure and energy under deformation. DNA in nucleosome core particle is described as an example. The structure and energy of nucleosomal DNA is calculated based on its sequence and positioning state. Our theory is on the level of base pair step parameters. A quadratic elastic energy function describes the deformational energy of the molecule in which sequence dependency appears in a matrix of coupling constants. This rigidity matrix has been evaluated based on molecular dynamics simulation data and X-ray crystallography structures of DNA. The inferred structure has been calculated through minimization of the energy and the results show remarkable similarity with X-ray data. Figure 1 compares the base pair step parameters of a typical nucleosomal DNA (NCP147: PDB id 1kx5) with its inferred structure. Although there is no sequence-specific interaction of bases and the histone core, we found considerable sequence dependency for nucleosomal DNA positioning. The wrapping affinity for 5S rRNA, 601 positioning, TATA box, TGGA repeat, and NCP147 sequences are calculated and compared with the experimental data. The structural energy differences, ΔΔG, are in good agreement with the data obtained by calorimetric approaches for natural sequences (Table 1). We argue that structural energy determines the natural state of nucleosomal DNA and is the main reason for affinity differences in vitro. This theory can be utilized for the DNA structure and energy determination in protein–DNA complexes in general.

Alkylation of duplex DNA in nucleosome core particles by duocarmycin SA and yatakemycin

(+)-Yatakemycin (1, Fig. 1) and (+)-duocarmycin SA (2) are exceptionally potent, naturally occurring antitumor agents that derive their biological properties through a characteristic sequence-selective DNA-alkylation reaction. Studies have shown that both the AT-rich binding selectivity (shape-selective recognition) and the alkylation catalysis (shape-dependent catalysis) that contribute to the alkylation selectivity are dependent on the DNA minor groove shape and size characteristics of an AT-rich sequence (ref. 6 and references therein; refs. 7,8). Here we report the alkylation properties of yatakemycin and duocarmycin SA on free DNA (alpha-satellite DNA) and the same sequence bound in a nucleosome core particle (NCP) modeling the state of DNA in eukaryotic cells. Both compounds showed a clear, relatively unaltered ability to alkylate DNA packaged in NCPs in terms of both alkylating efficiency and sequence selectivity, despite the steric and conformational perturbations imposed by NCP packaging. These findings highlight the dynamic nature of NCP-bound DNA and illustrate that cell- and protein-free DNA-alkylation studies of members of this class of antitumor drugs provide valuable insights into their properties.

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.

Use of the Transglutaminase Reaction To Study the Dissociation of Histone N-Terminal Tails from DNA in Nucleosome Core Particles

We have recently shown that core histones are glutaminyl substrates for transglutaminase (TGase) and that when native nucleosome cores are incubated with monodansylcadaverine (DNC) as donor amine, this fluorescent probe is incorporated into Gln5 and Gln19 of H3 and in Gln22 of H2B [Ballestar et al. (1996) J. Biol. Chem. 271, 18817-18825]. In the present paper, we report that the cause by which Gln22 of H2B is modified in nucleosomes but not in the free histone is the interaction of the region containing that glutamine with DNA. We have used the specificity of the TGase reaction to study the changes induced by increasing ionic strength in the interaction between the histone N-terminal tails and nucleosome DNA by two different approaches. First, the reactivity of the histone tail glutamines was employed to monitor changes in the interactions between the regions containing these residues and DNA. Second, by using reconstituted nucleosome core particles containing either H2B modified with DNC by the TGase reaction at Gln22 or H3 modified with the same procedure at Gln5 and Gln19, the dissociation of the histone tails was followed by the decrease of the fluorescence anisotropy of the probe. These methods allowed us to describe two ionic strength dependent structural transitions of the histone tails not reported to date. In the case of H2B, the first one occurs at very low ionic strength, and it can be assigned to an increase in the mobility of Gln22. The second one results in the cooperative release of a region of the tail that includes lysine residues next to Gln22, and it is followed by the overall release of the entire tail, described by other workers.

High Mobility Group Proteins 14 and 17 Can Prevent the Close Packing of Nucleosomes by Increasing the Strength of Protein Contacts in the Linker DNA

High mobility group (HMG) proteins 14 and 17 are abundant chromatin-associated proteins found in all higher eukaryotic nuclei. This observation demonstrates that HMGs 14 and 17 must have an important and universal function with regard to the structure and function of chromatin. What this function is, including how they interact with a nucleosomal array in vivo, is not known. Recently, we have demonstrated that HMGs 14 and 17 can organize nucleosomes into a regular array and increase the repeat length from 145 to about 160-165 base pairs in vitro. In addition, they can increase the apparent repeat length of chromatin deficient in histones H2A/H2B from 125 to approximately 145 base pairs. Importantly, this template was transcriptionally active. In this study, we report five new observations that begin to address the mechanism by which HMGs 14 and 17 space nucleosomal particles. First, we demonstrate that both human placenta HMG 14 and HMG 17 can space nucleosomes to produce a chromatin template with a repeat length around 160 base pairs. This result further highlights the similarity between these proteins in terms of protein structure and perhaps function. Second, we show that digestion of HMG containing chromatin with micrococcal nuclease produces DNA fragments that were approximately 10 and 20 base pairs longer than nucleosome core-particle DNA. This suggests that HMG 14 or HMG 17 can protect, directly or indirectly, at least an additional 10 base pairs of linker DNA from micrococcal digestion. However, this HMG-containing particle does not produce a strong kinetic block, and further digestion results in the eventual accumulation of DNA fragments 145 base pairs in length. Third, by comparing the full-length protein with different domains, we demonstrate that the acidic carboxyl-terminal domain is absolutely required for nucleosome spacing, neither the nucleosome binding domain of HMG 14 or HMG 17 nor the amino-terminal domain plus the nucleosome binding domain of HMG 14 could space nucleosomes. Fourth, we demonstrate that extensive micrococcal nuclease digestion of chromatin deficient in histones H2A/H2B led to the accumulation of DNA fragments about 110 base pairs in length, which is presumably the length of DNA associated with a nucleosomal particle deficient in one H2A/H2B dimer. Incorporation of either HMG 14 or HMG 17 into this chromatin results in the disappearance of this band and increase in the accumulation of fragments around 140-150 base pairs in length. Finally, in contrast to spacing of complete nucleosomes, we find that the nucleosome binding domain of HMG 17 (but not the nucleosome binding of HMG 14) is the only domain required for spacing of H2A/H2B-deficient chromatin.

Two new photoaffinity polyamines appear to alter the helical twist of DNA in nucleosome core particles

Two new photoaffinity derivatives of polyamines have been synthesized by the reaction of spermine or spermidine with methyl 4-azidobenzimidate. The new compounds were purified chromatographically and characterized by several methods including proton magnetic resonance spectroscopy. The spermine derivative is N1-ABA-spermine [(azidobenzamidino)spermine], and the spermidine derivative is a mixture of N1- and N8-ABA-spermidine. ABA-spermine stabilizes nucleosome core particles in thermal denaturation experiments, with similar but not identical effects when compared with the parent polyamine, spermine. In circular dichroism experiments, ABA-spermine was capable of producing a B----Z transition in poly(dG-m5dC) at a concentration of 30 microM, compared with 5 microM required to produce the same effect with spermine. On the other hand, ANB-spermine [(azidonitrobenzoyl)spermine; Morgan, J. E., Calkins, C. C., & Matthews, H. R. (1989) Biochemistry 28, 5095-5106] stabilized the B form of poly(dG-br5dC). ABA-spermine is a potent inhibitor of ornithine decarboxylase from Escherichia coli, giving 50% inhibition at 0.12 mM, while ANB-spermine is a modest inhibitor, comparable to spermine or spermidine. Under conditions of nitrogen-limited growth, yeast take up ABA-spermine and ABA-spermidine at approximately one-third to half the rate of spermidine or spermine. In contrast, ANB-spermine was not significantly taken up. The photoaffinity polyamines were used to photoaffinity label the DNA in nucleosome core particles, and the sites of labeling were determined by exonuclease protection. All photoaffinity reagents showed both nonspecific labeling and specific sites of higher occupancy. However, the positions of the sites varied: the ANB-spermine sites confirmed those previously reported (Morgan et al., 1989); the ABA-spermine and ABA-spermidine sites were spaced at 9.8 bp intervals from the 3' end of each DNA strand. This observation, together with the effect of spermine on the circular dichroism of DNA in nucleosome core particles, implies that polyamines alter the helical twist of DNA in nucleosome core particles. The ABA-polyamines are offered as general-purpose photoaffinity polyamine reagents.

Association of nucleosome core particle DNA with different histone oligomers

In non-denaturing low ionic strength gels, the titration of core DNA with H2A,H2B produces five well-defined bands. Quantitative densitometry and cross-linking experiments indicate that these bands are due to the successive binding of H2A,H2B dimers to core DNA. Only two bands are obtained with DNA-(H3,H4) samples. The slower of these bands is broad and presumably corresponds to two complexes containing one and two H3,H4 tetramers, respectively. In gels of higher ionic strength, DNA-(H2A,H2B) samples produce an ill-defined band, suggesting that the lifetime of the complexes containing H2A,H2B is relatively short. However, the low intensity of the free DNA band observed in these gels indicates that most of the DNA is associated with H2A,H2B. In agreement with this, our results obtained using different techniques (sedimentation, cross-linking, trypsin and nuclease digestions, and thermal denaturation) demonstrate that the association of H2A,H2B with core DNA occurs in free solution in both the absence and presence of NaCl (0.1 to 0.2 M). The low mobilities of DNA-(H2A,H2B) complexes, together with sedimentation and DNase I digestion results, indicate that the DNA in these complexes is not folded into the compact structure found in the core particle. Furthermore, non-denaturing gels have been used to study the dynamic properties of DNA-(H2A,H2B) and DNA-(H3,H4) complexes in 0.2 M-NaCl. Our results show that: (1) H2A,H2B and H3,H4 can associate, respectively, with DNA-(H3,H4) and DNA-(H2A,H2B) to produce complexes containing the four core histones; (2) DNA-(H2A,H2B) and DNA-(H3,H4) are able to transfer histones to free core DNA; (3) an exchange of histone pairs takes place between DNA-(H2A,H2B) and DNA-(H3,H4) and produces complexes with the same histone composition as that of the normal nucleosome core particle; and (4) although both histone pairs can exchange, histones H2A,H2B show a higher tendency than H3,H4 to migrate from one incomplete core particle to another. The complexes produced in these reactions have the same compact structure as reconstituted core particles containing the four core histones. Our kinetic results are consistent with a reaction mechanism in which the transfer of histones involves direct contacts between the reacting complexes. The possible participation of these spontaneous reactions on the mechanism of nucleosome assembly is discussed.

Stability of nucleosome placement in newly repaired regions of DNA.

Rearrangements of chromatin structure during excision repair of UV-damaged DNA appear to involve unfolding of nucleosomal DNA while repair is taking place, followed by refolding of this DNA into a native nucleosome structure. Recently, we found that repair patches are not distributed uniformly along the DNA in nucleosome core particles immediately following their refolding into nucleosomes (Lan, S. Y., and Smerdon, M. J. (1985) Biochemistry, 24,7771). Therefore, the distribution of repair patches in nucleosome core DNA was used to monitor the stability of nucleosome placement in these regions. Our results indicate that in nondividing human cells undergoing excision repair there is a slow change in the positioning of nucleosomes in newly repaired regions of chromatin, resulting in the eventual randomization of repair patches in nucleosome core DNA. Furthermore, the nonrandom placement of nucleosomes observed just after the refolding event is not re-established during DNA replication. Possible mechanisms for this change in nucleosome placement along the DNA are discussed.

Nucleosome rearrangement in vitro. 2. Formation of nucleosomes in newly repaired regions of DNA.

We have reported previously that immediately following nucleotide excision repair in human cells the newly repaired DNA lacks a nucleosome conformation [Smerdon, M. J., & Lieberman, M. W. (1980) Biochemistry 19, 2992-3000]. In this study, we have examined the ability of these nascent DNA regions to acquire a nucleosome structure in vitro by incubating intact or H1-depleted nuclei in buffers containing different salt concentrations (0.025-0.625 M KCl) at 0 or 37 degrees C. Nucleosomes were detected in these regions by an increase in the level of repair-incorporated nucleotides associated with isolated nucleosome core particle DNA. Our results indicate that the nascent DNA is resistant to nucleosome formation during the low-salt transition where the limiting repeat length decreases from approximately 190 to 168 base pairs (bp) [Watkins, J. F., & Smerdon, M. J. (1985) Biochemistry (preceding paper in this issue)]. This result provides further evidence that the nascent DNA is indeed in a nonnucleosomal state. At higher salt concentrations (greater than 0.4 M), where the nucleosome repeat length decreases to a limiting value of approximately 146 bp, there was an increase in nucleosome formation in nascent DNA that correlated with the decrease in limiting repeat length. However, we did not observe a complete randomization of the repair-incorporated nucleotides. Indeed, even at the highest salt concentration used (0.625 M), we never observed more than 50% of the nascent DNA associated with the isolated core particles. This was the case even though a major portion of the nucleosomes had a limiting value repeat length following the high-salt incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Another potential artifact in the study of nucleosome phasing by chromatin digestion with micrococcal nuclease

We show that, contrary to expectations, restriction enzyme cleavage of chicken erythrocyte nucleosome core particle DNA generates a series of distinct subnucleosome fragments. These fragments do not result from bulk nucleosome phasing in vivo, but arise from micrococcal nuclease cleavages internal to the core particle, at roughly 10-base pair intervals and at AT-rich sequences. Those 145-base pair DNA fragments remaining intact are a biased population in which the guanine content can fluctuate by as much as 10%, with a 10-base pair period. We suggest that these same considerations, when applied to a unique DNA sequence, are the true explanation for several previous claims for nucleosome phasing.

The binding of a carcinogen to the nucleosomal and non-nucleosomal regions of the simian virus 40 chromosome in vivo.

The effect of chromatin structure on the binding of a chemical carcinogen to the genomic DNA was studied. The binding in vivo of the ultimate carcinogen, benzo-pyrene 7,8,-diol,-9,10-epoxide, to various regions of the SV40 chromosome was revealed by an immunological method. Particular attention was given to restriction fragments which include the origin of replication which is "non-nucleosomal" in a significant fraction of the chromosomes. The distribution of (+/-) trans-7,8-dihydrobenzo[alpha]pyrene-7,8-diol-9,10-epoxide (BPDE) adducts was studied in 1) SV40 DNA modified in vitro to a level of 20 adducts/molecule, 2) DNA from SV40 chromosomes modified in vivo to a level of less than 1 adduct, and 3) DNA from only those chromosomes with an open origin of replication. In other experiments, the binding of BPDE to the origin region was compared to the binding to nucleosome core particle DNA from the viral chromosome. The origin region bound 1.7-fold more BPDE than core DNA, while linker DNA is 3-fold more modified than core DNA. However, the origin region was only about 20% more modified than any other region of the chromosome. We conclude that while the conformation of the DNA in chromatin has a slight effect on its accessibility to the carcinogen, the SV40 chromosome does not contain a particular "hot spot" which is preferentially modified by BPDE.

Dynamics of DNA in chromatin and DNA binding mode to core protein.

We have studied the dynamics of DNA in nucleosome core particles and in the linker region of chromatin using nanosecond fluorescence anisotropy decay measurements of intercalated ethidium. DNA in the core undergoes torsional motions to the same extent as the linker DNA in extended chromatin. We therefore concluded that the binding of DNA to the histone octamer is relatively weak or limited to a few points; stretches of at least several tens of base pairs exist which can move as freely as DNA in solution.

Convenient continuous sonication method for the preparation of medium-sized polydeoxynucleotides

We recently reported results on polydeoxynucleotides prepared for 31p NMR studies by sonication [1]. For these studies we required samples of medium length, from ca. 50-200 base pairs (bp), comparable to the size of DNA in nucleosome core particles. To obtain this size range we utilized the continuous low-temperature sonication procedure described for calf-thymus DNA by Davis and Phillips [2]. We have extended this procedure to small volumes of synthetic polydeoxynucleotides in a convenient preparative method. The apparatus used is shown in Fig. 1. The temperature was continuously monitored and kept between 0-4°C using a circulating cooling bath. Volumes down to 1 ml are possible in this apparatus, although we generally used 2-4 ml containing 5-100 A260n m units in 0.1-1.0M NaCI solution. A tapered microprobe was used with a He~t Systems-Ultrasonics W225R sonicator. The power level was optimized just below observable cavitation-bubbling (ca. 25 W). Experiments comparing the efficiency of breakage using the pulsed capability for comparable time periods (e.g. 50% duty cycle for twice the total time) indicated that continuous sonication was more efficient. The size distribution of the samples after sonication was determined by gel electrophoresis. Following development of the gel in ethidium bromide, the photographic negatives were scanned with a microdensitometer (Joyce-Loebl double beam, model IIIB) and the scans were digitized. The distances migrated were converted to base pairs relative to known standards using the formula described by Kovacic and Van Holde [3]. Results for sonication for different time periods showed that it was possible to obtain fairly well defined distributions with several base sequences [1]. For example, 10 min sonication of poly(dGdC), poly(dGdC) gave a maximum at

Stability of the primary organization of nucleosome core particles upon some conformational transitions.

The sequential arrangement of histones along DNA in nucleosome core particles was determined between 0.5 and 600 mM salt and from 0 to 8 M urea. These concentrations of salt and urea up to 6 M had no significant effect on the linear order of histones along DNA but 8 M urea caused the rearrangement of histones. Conformational changes in cores have been identified within these ranges of conditions by several laboratories 8-21. Also, abrupt structural changes in the cores, apparently their unfolding, were found by gel electrophoresis to occur at urea concentration, between 4 and 5 M. 600 mM salt and 6 M urea were shown to relax the binding of histones to DNA in cores but do not however release histones or some part of their molecules from DNA. It appears therefore that nucleosomal cores can undergo some conformational transitions and unfolding whereas their primary organization remains essentially unaffected. These results are consistent with a model of the core particles in which the histone octamer forms something like a helical "rim" along the superhelical DNA and histone-histone interactions beyond the "rim" are rather weak in comparison with those within the "rim".

31P-NMR studies of DNA in nucleosome core particles.

Abstract31P Nmr parameters (δ, T1, W1/2, and NOE) were measured for the DNA in nucleosome core particles at three frequencies and compared with similar data for the histone‐free DNA. An essentially linear relationship was found between the frequency of observation and line‐width for the single broad envelope of 31P resonances of the DNA in the nucleosome cores. We attributed this largely to chemical shift dispersion, with smaller contributions from chemical shift anisotropy and dipolar broadening. These results suggest the presence of different environments for phosphorus atoms in the core particles. However, within the accuracy of the method, no asymmetry in the resonance could be detected, which would tend to rule out any significant degree of DNA “kinking.” To investigate the interactions of the DNA and histones within the core particles we also studied transitions induced by urea and by temperature. Urea caused two stepwise increases in linewidth, which we attributed to conformational changes. A biphasic transition was also observed in the temperature profile, consistent with previous optical studies [Weischet et. al. (1978) Nucleic Acids Res. 5, 139]. Various models with different types of local mobility were examined by the relaxation theory. A model of isotropic motion having a broad distribution of correlation times gave a fairly good fit to the 31P‐nmr data.

Asymmetric lateral distribution of unshielded phosphate groups in nucleosomal DNA and its role in DNA bending.

We suggest that an asymmetric charge neutralization of DNA phosphate groups produces part of the driving force for nucleosome folding. In nucleosome core particle DNA, many of the phosphate groups are neutralized by histones, and a lateral alignment of these histones along the core DNA has been demonstrated [Mirzabekov A. D., Shick, V. V., Belyavsky, A. V. & Bavykin, S. G. (1978) Proc. Natl. Acad. Sci. USA 75, 4184--4189]. Histones appear to shield DNA phosphatases asymmetrically at one side of the surface of the DNA double helix along all its length inside the core. The external side of the DNA helix remains unneutralized. The electrostatic repulsion between negatively charged unneutralized phosphates may fold the nucleosomal DNA towards the side occupied by histones.

1H NMR investigation of the conformational states of DNA in nucleosome core particles.

In this study 1H NMR has been used to investigate the conformational state of DNA in nucleosome core particles. The nucleosome core particles exhibit partially resolved low field (10-15 ppm) spectra due to imino protons in Watson-Crick base pairs (one resonance per GC or AT base pair). To a first approximation, the spectrum is virtually identical with that of protein-free 140 base pair DNA, and from this observation we draw two important conclusions: (i) Since the low field spectra of DNA are known to be sensitive to conformation, the conformation of DNA in the core particles is essentially the same as that of free DNA (presumably B-form), (ii) since kinks occurring at a frequency at 1 in 10 or 1 in 20 base pairs would result in a core particle spectrum different from that of free DNA we find no NMR evidence supporting either the Crick-Klug or the Sobell models for kinking DNA around the core histones. Linewidth considerations indicate that the rotational correlation time for the core particles is approximately 1.5 X 10(-7) sec, whereas the end-over-end tumbling time of the free 140 base pair DNA is 3 X 10(-7) sec.