Nucleotide excision repair from site-specifically platinum-modified nucleosomes

Nucleotide excision repair is a major cellular defense mechanism against the toxic effects of the anticancer drug cisplatin and other platinum-based chemotherapeutic agents. In this study, mononucleosomes were prepared containing either a site-specific cis-diammineplatinum(II)-DNA intrastrand d(GpG) or a d(GpTpG) cross-link. The ability of the histone core to modulate the excision of these defined platinum adducts was investigated as a model for exploring the cellular response to platinum-DNA adducts in chromatin. Comparison of the extent of repair by mammalian cell extracts of free and nucleosomal DNA containing the same platinum-DNA adduct reveals that the nucleosome significantly inhibits nucleotide excision repair. With the GTG-Pt DNA substrate, the nucleosome inhibits excision to about 10% of the level observed with free DNA, whereas with the less efficient GG-Pt DNA substrate the nucleosome inhibited excision to about 30% of the level observed with free DNA. The effects of post-translational modification of histones on excision of platinum damage from nucleosomes were investigated by comparing native and recombinant nucleosomes containing the same intrastrand d(GpTpG) cross-link. Excision from native nucleosomal DNA is approximately 2-fold higher than the level observed with recombinant material. This result reveals that post-translational modification of histones can modulate nucleotide excision repair from damaged chromatin. The in vitro system established in this study will facilitate the investigation of platinum-DNA damage by DNA repair processes and help elucidate the role of specific post-translational modification in NER of platinum-DNA adducts at the physiologically relevant nucleosome level.     

The histone octamer influences the wrapping direction of DNA on it: Brownian dynamics simulation of the nucleosome chirality

In eukaryote nucleosome, DNA wraps around a histone octamer in a left-handed way. We study the process of chirality formation of nucleosome with Brownian dynamics simulation. We model the histone octamer with a quantitatively adjustable chirality: left-handed, right-handed or non-chiral, and simulate the dynamical wrapping process of a DNA molecule on it. We find that the chirality of a nucleosome formed is strongly dependent on that of the histone octamer, and different chiralities of the histone octamer induce its different rotation directions in the wrapping process of DNA. In addition, a very weak chirality of the histone octamer is quite enough for sustaining the correct chirality of the nucleosome formed. We also show that the chirality of a nucleosome may be broken at elevated temperature.     

体外装配核小体过程组蛋白浓度依赖的动力学模型

为探索组蛋白浓度对核小体体外装配的影响,本研究表达纯化了4种组蛋白,通过控制实验反应体系中组蛋白的浓度,利用盐透析法在体外装配了核小体,检测分析了组蛋白浓度与核小体组装效率的关系。以此实验数据为基础,提出了核小体组装过程组蛋白浓度依赖性的动力学模型。实验结果发现,反应体系中组蛋白浓度与核小体生成量呈典型的线性关系。依据动力学理论模型,进行线性回归拟合,回归系数达到0.963;经计算601 DNA序列组装核小体的反应速率常数k为1.49×10^-5mL·h·μg^-1。CS1序列验证动力学模型的线性回归相关系数为0.989,反应速率常数为1.52×10^-5mL·h·μg^-1。该实验方法及动力学模型中反应速率常数k可用于评价相同长度的DNA序列组装核小体的能力、组蛋白与其突变体以及组蛋白变体之间形成核小体结构能力的差异。该动力学模型的建立为理解核小体装配、核小体定位、染色质结构等相关问题提供了理论指导。     

Solution structure of variant H2A.Z.1 nucleosome investigated by small-angle X-ray and neutron scatterings

Solution structures of nucleosomes containing a human histone variant, H2A.Z.1, were measured by small-angle X-ray and neutron scatterings (SAXS and SANS). SAXS revealed that the outer shape, reflecting the DNA shape, of the H2A.Z.1 nucleosome is almost the same as that of the canonical H2A nucleosome. In contrast, SANS employing a contrast variation technique revealed that the histone octamer of the H2A.Z.1 nucleosome is smaller than that of the canonical nucleosome. The DNA within the H2A.Z.1 nucleosome was more susceptible to micrococcal nuclease than that within the canonical nucleosome. These results suggested that the DNA is loosely wrapped around the histone core in the H2A.Z.1 nucleosome.     

New histone supply regulates replication fork speed and PCNA unloading

Correct duplication of DNA sequence and its organization into chromatin is central to genome function and stability. However, it remains unclear how cells coordinate DNA synthesis with provision of new histones for chromatin assembly to ensure chromosomal stability. In this paper, we show that replication fork speed is dependent on new histone supply and efficient nucleosome assembly. Inhibition of canonical histone biosynthesis impaired replication fork progression and reduced nucleosome occupancy on newly synthesized DNA. Replication forks initially remained stable without activation of conventional checkpoints, although prolonged histone deficiency generated DNA damage. PCNA accumulated on newly synthesized DNA in cells lacking new histones, possibly to maintain opportunity for CAF-1 recruitment and nucleosome assembly. Consistent with this, in vitro and in vivo analysis showed that PCNA unloading is delayed in the absence of nucleosome assembly. We propose that coupling of fork speed and PCNA unloading to nucleosome assembly provides a simple mechanism to adjust DNA replication and maintain chromatin integrity during transient histone shortage.     

Biochemical characterization of the placeholder nucleosome for DNA hypomethylation maintenance

DNA methylation functions as a prominent epigenetic mark, and its patterns are transmitted to the genomes of offspring. The nucleosome containing the histone H2A.Z variant and histone H3K4 mono-methylation acts as a “placeholder” nucleosome for DNA hypomethylation maintenance in zebrafish embryonic cells. However, the mechanism by which DNA methylation is deterred by the placeholder nucleosome is poorly understood. In the present study, we reconstituted the placeholder nucleosome containing histones H2A.Z and H3 with the Lys4 mono-methylation. The thermal stability assay revealed that the placeholder nucleosome is less stable than the canonical nucleosome. Nuclease susceptibility assays suggested that the nucleosomal DNA ends of the placeholder nucleosome are more accessible than those of the canonical nucleosome. These characteristics of the placeholder nucleosome are quite similar to those of the H2A.Z nucleosome without H3K4 methylation. Importantly, the linker histone H1, which is reportedly involved in the recruitment of DNA methyltransferases, efficiently binds to all of the placeholder, H2A.Z, and canonical nucleosomes. Therefore, the characteristics of the H2A.Z nucleosome are conserved in the placeholder nucleosome without synergistic effects on the H3K4 mono-methylation.     

Chromatin assembled in the presence of cytosine arabinoside has a short nucleosome repeat.

Incubation of MSB cells with cytosine arabinoside (1-beta-D-arabinofuranosylcytosine, ara-C) inhibits 3H-thymidine incorporation into nascent DNA while nucleosome core histone synthesis proceeds in molar stoichiometry at about 20% of control rates. The excess nascent histone is incorporated into chromatin and nucleosome cores are assembled normally on the small amount of DNA which is synthesized at submaximal levels of ara-C. This DNA becomes packaged into a shortened nucleosome repeat, however. These results indicate that the nucleosome core is a strongly conserved unit of chromatin replication and suggest that the stoichiometry of nascent histone to DNA may be one factor influencing the establishment of the nucleosome repeat length. It cannot be the only factor, however, since the closely packed nucleosomes made in the presence of ara-C begin to return to their normal spacing within six hours after reversal.     

Influence of DNA topology and histone tails in nucleosome organization on pBR322 DNA.

Recently, we have found that the assembly of nucleosomes reconstituted on negatively supercoiled DNA is cooperative. In the present paper the role of DNA topology and of histone tails in nucleosome assembly was explored. Reconstituted minichromosomes on relaxed DNA at different histone/DNA ratios (R) were assayed by topological analysis and electron microscopy visualization. Both methods show a linear relationship between average nucleosome number (N) and R. This suggests that in the case of relaxed DNA, cooperative internucleosomal interactions are small or absent. The influence of histone tails in nucleosome assembly was studied on minichromosomes reconstituted with trypsinized histone octamer on negatively supercoiled DNA by topological analysis. The topoisomers distribution, after trypsinization, dramatically changes, indicating that nucleosome-nucleosome interactions are remarkably decreased. These results show that, in chromatin folding, in addition to the well known role of histone H1, the interactions between histone octamer tails and DNA are also of importance.     

SWI/SNF unwraps,slides, and rewraps the nucleosome

The structure of the SWI/SNF-remodeled nucleosome was characterized with single base-pair resolution by mapping the contacts of specific histone fold residues with nucleosomal DNA. We demonstrate that SWI/SNF peels up to 50 bp of DNA from the edge of the nucleosome, translocates the histone octamer beyond the DNA ends via a DNA bulge propagation mechanism, and promotes the formation of an intramolecular DNA loop between the nucleosomal entry and exit sites. This stable altered nucleosome conformation also exhibits alterations in the distance between contacts of specific histone residues with DNA and higher electrophoretic and sedimentation mobility, consistent with a more compact molecular shape. SWI/SNF converts a nucleosome to the altered state in less than 1 s, hydrolyzing fewer than 10 ATPs per event.     

DNA sequence-dependent contributions of core histone tails to nucleosome stability: differential effects of acetylation and proteolytic tail removal

Modulation of nucleosome stability in chromatin plays an important role in eukaryotic gene expression. The core histone N-terminal tail domains are believed to modulate the stability of wrapping nucleosomal DNA and the stability of the chromatin filament. We analyzed the contribution of the tail domains to the stability of nucleosomes containing selected DNA sequences that are intrinsically straight, curved, flexible, or inflexible. We find that the presence of the histone tail domains stabilizes nucleosomes containing DNA sequences that are intrinsically straight or curved. However, the tails do not significantly contribute to the free energy of nucleosome formation with flexible DNA. Interestingly, hyperacetylation of the core histone tail domains does not recapitulate the effect of tail removal by limited proteolysis with regard to nucleosome stability. We find that acetylation of the tails has the same minor effect on nucleosome stability for all the selected DNA sequences. A comparison of histone partitioning between long donor chromatin, acceptor DNA, and free histones in solution shows that the core histone tails mediate internucleosomal interactions within an H1-depleted chromatin fiber amounting to an average free energy of about 1 kcal/mol. Thus, such interactions would be significant with regard to the free energies of sequence-dependent nucleosome positioning. Last, we analyzed the contribution of the H2A/H2B dimers to nucleosome stability. We find that the intact nucleosome is stabilized by 900 cal/mol by the presence of the dimers regardless of sequence. The biological implications of these observations are discussed.     

Chromatin assembly: a basic recipe with various flavours

Packaging of eukaryotic genomes into chromatin is a hierarchical mechanism, starting with histone deposition onto DNA to produce nucleosome arrays, which then further fold and ultimately form functional domains. Recent studies provide interesting insight into how nucleosome assembly is coordinated with histone and DNA metabolism and underline the combined contribution of histone chaperones and chromatin remodelers. How these factors operate at a molecular level is a matter of current investigation. New data highlight the importance of histone dimers as deposition entities for de novo nucleosome assembly and identify dedicated machineries involved in histone variant deposition.     

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The coordinated process of DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3-H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three histone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3-H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3-H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.     

Histone tail network and modulation in a nucleosome

The structural unit of eukaryotic chromatin is a nucleosome, comprising two histone H2A/H2B heterodimers and one histone (H3/H4)₂ tetramer, wrapped around by ∼146-bp core DNA and linker DNA. Flexible histone tails sticking out from the core undergo posttranslational modifications that are responsible for various epigenetic functions. Recently, the functional dynamics of histone tails and their modulation within the nucleosome and nucleosomal complexes have been investigated by integrating NMR, molecular dynamics simulations, and cryo-electron microscopy approaches. In particular, recent NMR studies have revealed correlations in the structures of histone N-terminal tails between H2A and H2B, as well as between H3 and H4 depending on linker DNA, suggesting that histone tail networks exist even within the nucleosome.     

Effect of trypsinization and histone H5 addition on DNA twist and topology in reconstituted minichromosomes.

Free DNA in solution exhibits an untwisting of the double helix with increasing temperature. We have shown previously that when DNA is reconstituted with histones to form nucleosome core particles, both the core DNA and the adjacent linker DNA are constrained from thermal untwisting. The origin of this constraint is unknown. Here we examine the effect of two modifications of nucleosome structure on the constraint against thermal untwisting, and also on DNA topology. In one experiment, we removed the highly positively charged histone amino and carboxy termini by trypsinization. Alternatively, we added histone H5, a histone H1 variant from chick erythrocytes. Neither of these modifications had any major effect on DNA topology or twist in the nucleosome.     

Uracil DNA Glycosylase Activity on Nucleosomal DNA Depends on Rotational Orientation of Targets

The activity of uracil DNA glycosylases (UDGs), which recognize and excise uracil bases from DNA, has been well characterized on naked DNA substrates but less is known about activity in chromatin. We therefore prepared a set of model nucleosome substrates in which single thymidine residues were replaced with uracil at specific locations and a second set of nucleosomes in which uracils were randomly substituted for all thymidines. We found that UDG efficiently removes uracil from internal locations in the nucleosome where the DNA backbone is oriented away from the surface of the histone octamer, without significant disruption of histone-DNA interactions. However, uracils at sites oriented toward the histone octamer surface were excised at much slower rates, consistent with a mechanism requiring spontaneous DNA unwrapping from the nucleosome. In contrast to the nucleosome core, UDG activity on DNA outside the core DNA region was similar to that of naked DNA. Association of linker histone reduced activity of UDG at selected sites near where the globular domain of H1 is proposed to bind to the nucleosome as well as within the extra-core DNA. Our results indicate that some sites within the nucleosome core and the extra-core (linker) DNA regions represent hot spots for repair that could influence critical biological processes.     

Computational analysis suggests a highly bendable, fragile structure for nucleosomal DNA

Eukaryotic chromosomal DNA coils around histones to form nucleosomes. Although histone affinity for DNA depends on DNA sequence patterns, how nucleosome positioning is determined by them remains unknown. Here, we show relationships between nucleosome positioning and two structural characteristics of DNA conferred by DNA sequence. Analysis of bendability and hydroxyl radical cleavage intensity of nucleosomal DNA sequences indicated that nucleosomal DNA is bendable and fragile and that nucleosome positional stability was correlated with characteristics of DNA. This result explains how histone positioning is partially determined by nucleosomal DNA structure, illuminating the optimization of chromosomal DNA packaging that controls cellular dynamics.     

Chromatin assembly on plasmid DNA in vitro. Apparent spreading of nucleosome alignment from one region of pBR327 by histone H5.

We have found that histone H5 (or H1) induces physiological nucleosome spacings and extensive ordering on some plasmid constructions, but not on others, in a fully defined in vitro system. Plasmid pBR327 containing DNA insertions with lengths close to 300 base-pairs permitted histone H5 to induce a remarkable degree of nucleosome alignment. Seventeen multiples of a unit 210(+/- 4) base-pair repeat, covering the entire plasmid, were detected. Plasmid pBR327, not containing a DNA insert, permitted continuous alignment of only a few nucleosomes. These observations suggest that a necessary requirement in this system for histone H5 (or H1)-induced nucleosome alignment on small (less than 4 kb; 1 kb = 10(3) bases or base-pairs) circular plasmids may be that the total DNA length must be close to an integer multiple of the nucleosome repeat length generated, a type of boundary effect. Consistent with this hypothesis, five deletion constructs of pBR327 (not containing inserts), that spanned 64% of the plasmid, and possessed DNA lengths close to integer multiples of 210 base-pairs, permitted nucleosome alignment by histone H5. We have also found that plasmid length adjustment is not a sufficient condition for nucleosome alignment. For example, plasmids pBR322 and pUC18 did not permit nucleosome alignment when adjusted to near-integer multiples of 210 base-pairs. Also, for pBR327 that contained a length-adjusted deletion in one particular region, appreciable nucleosome alignment no longer occurred. These data suggest that a contiguous approximately 800 base-pair region of pBR327, interrupted in pBR322 and not present in pUC18, can nucleate histone H5-induced nucleosome alignment, which can then spread to adjacent chromatin. Supporting this idea, a positioned five-nucleosome array appears to originate in the required region. Additionally, on a larger (6.9 kb) plasmid construction, the "chromatin organizing region" of pBR327 and adjacent DNA on one side of it exhibited preferred H5-induced nucleosome alignment.