NAP1-Assisted Nucleosome Assembly on DNA Measured in Real Time by Single-Molecule Magnetic Tweezers

While many proteins are involved in the assembly and (re)positioning of nucleosomes, the dynamics of protein-assisted nucleosome formation are not well understood. We study NAP1 (nucleosome assembly protein 1) assisted nucleosome formation at the single-molecule level using magnetic tweezers. This method allows to apply a well-defined stretching force and supercoiling density to a single DNA molecule, and to study in real time the change in linking number, stiffness and length of the DNA during nucleosome formation. We observe a decrease in end-to-end length when NAP1 and core histones (CH) are added to the dsDNA. We characterize the formation of complete nucleosomes by measuring the change in linking number of DNA, which is induced by the NAP1-assisted nucleosome assembly, and which does not occur for non-nucleosomal bound histones H3 and H4. By rotating the magnets, the supercoils formed upon nucleosome assembly are removed and the number of assembled nucleosomes can be counted. We find that the compaction of DNA at low force is about 56 nm per assembled nucleosome. The number of compaction steps and associated change in linking number indicate that NAP1-assisted nucleosome assembly is a two-step process.     

In vitro chromatin assembly promoted by the Xenopus laevis S-150 cell-free extract is enhanced by treatment with RNase A.

Cell-free extracts employed as chromatin assembly systems contain a myriad of proteins, polyanions and nucleic acids. The roles of ATP, MgCl2 and other cofactors in the catalysis of nucleosome formation by the Xenopus laevis oocyte S-150 have yet to be established unequivocally. In this study we examine the influence of RNA in the assembly process. Under reaction conditions that inhibit nucleosome formation (+ EDTA), pretreatment of the extract with RNase A revives the chromatin assembly machinery while the rate of DNA supercoiling is stimulated significantly. Addition of purified RNA blocks DNA supercoiling. Taken together, these data suggest that the parameters surrounding in vitro chromatin assembly are variable and subject to modulation by endogenous factors.     

Chromatin reconstitution on small DNA rings. IV. DNA supercoiling and nucleosome sequence preference.

Nucleosome formation on inverted repeats or on some alternations of purines and pyrimidines can be inhibited in vitro by DNA supercoiling through their supercoiling-induced structural transitions to cruciforms or Z-form DNA, respectively. We report here, as a result of study of single nucleosome reconstitutions on a DNA minicircle, that a physiological level of DNA supercoiling can also enhance nucleosome sequence preference. The 357 base-pair minicircle was composed of a promoter of phage SP6 RNA polymerase joined to a 256 base-pair fragment containing a sea urchin 5 S RNA gene. Nucleosome formation on the promoter was found to be enhanced on a topoisomer with in vivo superhelix density when compared to topoisomers of lower or higher superhelical densities, to the nicked circle, or to the linear DNA. In contrast, nucleosomes at other positions appeared to be insensitive to supercoiling. This observation relied on a novel procedure for the investigation of nucleosome positioning. The reconstituted circular chromatin was first linearized using a restriction endonuclease, and the linear chromatin so obtained was electrophoresed as nucleoprotein in a polyacrylamide gel. The gel showed well-fractionated bands whose mobilities were a V-like function of nucleosome positions, with the nucleosome near the middle migrating less. This behavior is similar to that previously observed for complexes of sequence-specific DNA-bending proteins with circularly permuted DNA fragments, and presumably reflects the change in the direction of the DNA axis between the entrance and the exit of the particle. Possible mechanisms for such supercoiling-induced modulation of nucleosome formation are discussed in the light of the supercoiling-dependent susceptibility to cleavage of the naked minicircle with S1 and Bal31 nucleases; and a comparison between DNase I cleavage patterns of the modulated nucleosome and of another, non-modulated, overlapping nucleosome.     

Assembly of nucleosomal DNA in a cell-free extract from wild-type and top1− strains ofUstilago maydis

An in vitro nucleosome assembly system has been established from cell-free extracts of the fungusUstilago maydis. The extract catalyzed DNA supercoiling in the absence of exogenously added co-factors such as ATP and MgCl₂ and was inhibited by moderate concentrations (200 mM) of KCl or NaCl. DNA supercoiling occurs via the formation of nucleosomes. Similar extracts, displaying the same activity, were prepared fromSaccharomyces cerevisiae andCandida albicans, suggesting that the extract preparation protocol may be useful for many lower eukaryotic systems. An extract prepared from a strain ofU. maydis lacking topoisomerase I failed to catalyze nucleosome assembly, clearly implicating this enzyme in this process. Addition of purified topoisomerase I, and, to a lesser extent, topoisomerase II, to the top1^? extract regenerated the supercoiling activity. Our results provide a method for preparing assembly extracts from organisms, that are particularly amenable to genetic manipulation.     

The Mu transposase interwraps distant DNA sites within a functional transpososome in the absence of DNA supercoiling

A Mu transpososome assembled on negatively supercoiled DNA traps five supercoils by intertwining the left (L) and right (R) ends of Mu with an enhancer element (E). To investigate the contribution of DNA supercoiling to this elaborate synapse in which E and L cross once, E and R twice, and L and R twice, we have analyzed DNA crossings in a transpososome assembled on nicked substrates under conditions that bypass the supercoiling requirement for transposition. We find that the transposase MuA can recreate an essentially similar topology on nicked substrates, interwrapping both E-R and L-R twice but being unable to generate the single E-L crossing. In addition, we deduce that the functional MuA tetramer must contribute to three of the four observed crossings and, thus, to restraining the enhancer within the complex. We discuss the contribution of both MuA and DNA supercoiling to the 5-noded Mu synapse built at the 3-way junction.     

Assembly of nucleosomal DNA in a cell-free extract from wild-type and top1? strains ofUstilago maydis

An in vitro nucleosome assembly system has been established from cell-free extracts of the fungusUstilago maydis. The extract catalyzed DNA supercoiling in the absence of exogenously added co-factors such as ATP and MgCl₂ and was inhibited by moderate concentrations (200 mM) of KCl or NaCl. DNA supercoiling occurs via the formation of nucleosomes. Similar extracts, displaying the same activity, were prepared fromSaccharomyces cerevisiae andCandida albicans, suggesting that the extract preparation protocol may be useful for many lower eukaryotic systems. An extract prepared from a strain ofU. maydis lacking topoisomerase I failed to catalyze nucleosome assembly, clearly implicating this enzyme in this process. Addition of purified topoisomerase I, and, to a lesser extent, topoisomerase II, to the top1^– extract regenerated the supercoiling activity. Our results provide a method for preparing assembly extracts from organisms, that are particularly amenable to genetic manipulation.     

Histone H1 preferentially binds to superhelical DNA molecules of higher compaction.

In chromatin, the physiological amount of H1 is one molecule per nucleosome or, roughly, one molecule per 200 bp of DNA. We observed that at such a stoichiometry, H1 selectively binds to supercoiled DNA with magnitude of sigma > or = 0.012 (both negative and positive), leaving relaxed, linear, or nicked DNA molecules unbound. When negative and positive DNA topoisomers of varying superhelicity are simultaneously present in the binding mixture, H1 selectively binds to the molecules with highest superhelicity; less supercoiled forms are gradually involved in binding upon increasing the amount of input protein. We explain this topological preference of H1 as the consequence of an increased probability for more than one H1-DNA contact provided by the supercoiling. The existence of simultaneous contacts of H1 with both intertwined DNA strands in the supercoiled DNA molecules is also inferred by topoisomerase relaxation of H1-DNA complexes that had been prefixed with glutaraldehyde.     

Negative constrained DNA supercoiling in archaeal nucleosomes

Archaeal histones have significant sequence and structural similarity to their eukaryal counterparts. However, whereas DNA is wrapped in negatively constrained supercoils in eukaryal nucleosomes, it has been reported that DNA is positively supercoiled by archaeal nucleosomes. This was inferred from experiments performed at low temperature and low salt concentrations, conditions markedly different from those expected for many archaea in vivo. Here, we report that the archaeal histones HMf and HTz wrap DNA in negatively constrained supercoils in buffers containing potassium glutamate (K-Glu) above 300 mM, either at 37 degrees C or at 70 degrees C. This suggests that high salt concentrations allow an alternate archaeal nucleosome topology: a left-handed tetramer rather than the right-handed tetramer seen in low salt conditions. In contrast, the archaeal histone MkaH produces DNA negative supercoiling at all salt concentrations, suggesting that this duality of structure is not possible for this atypical protein, which is formed by the association of two histone folds in a single polypeptide. These results extend the already remarkable similarity between archaeal and eukaryal nucleosomes, as it has been recently shown that DNA can be wrapped into either positive or negative supercoils around the H3/H4 tetramer. Negative supercoiling could correspond to the predominant physiological mode of DNA supercoiling in archaeal nucleosomes.     

Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity.

Undiluted extracts from eggs or oocytes of Xenopus laevis support the assembly of chromatin with physiologically spaced nucleosomes. Micrococcal nuclease and DNase I digestion experiments show that nucleosome formation as well as supercoiling of circular DNA concomitant to assembly do not require ATP or Mg2+. However these factors are essential for the stability and the physiological spacing of the assembled chromatin. gamma-S-ATP can substitute for ATP in this process. With topoisomers of defined linking number topological interconversions proceed by steps of unity, both in vitro as well as in vivo, indicating that topoisomerase I is dominantly acting in this process. Novobiocin sensitivity occurred only with diluted extracts and was unrelated to an inhibition of topoisomerase II. Finally, nucleosome assembly occurs efficiently on linear DNA although the assembled DNA is less stable than with circular DNA. From these results we propose that mature chromatin is formed in a two-step reaction. In the first step, nucleosome deposition occurs independently of ATP and Mg2+. Thus, nucleosome formation can be uncoupled from their spacing. In this step, topoisomerase activity is involved in the relaxation of the topological constraints generated by chromatin assembly rather than in the process of assembly itself. The second step, requiring ATP and Mg2+, generates properly spaced chromatin.     

Introduction of superhelical turns into DNA by adenoviral core proteins and chromatin assembly factors.

The interaction in vitro between adenoviral histone-like proteins and DNA in the presence of chromatin assembly factors was investigated. Viral core protein VII or its precursor pVII was incubated with DNA in the presence of an extract of HeLa cell chromatin, which mediates nucleosome assembly from histones and DNA. We have demonstrated that either protein can introduce superhelical turns into relaxed closed-circular DNA and that the presence of chromatin extract is necessary for the supertwisting effect. A greater density of superhelical turns was produced by pVII than by VII, but neither protein-DNA interaction resulted in the "physiological" amount of supertwisting produced by histones. The inhibition of histone-induced supercoiling by both proteins and the protection of turns in supertwisted starting material are also described. The nucleosome assembly factor, nucleoplasmin, fails to mediate the introduction of superhelical turns by VII or pVII.     

Supercoiled induced transition to the Z-DNA conformation affects the ability of a d(CG/GC)12 sequence to be organized into nucleosome-cores.

Nucleosome-cores were reconstituted by the salt-dialysis method onto closed circular pDHg16 DNA which contains a d(CG/GC)12 sequence. Alternating d(CG/GC)n sequences form left-handed Z-DNA readily when contained in negatively supercoiled DNA. We have investigated the ability of the d(CG/GC)12 sequence to be organized into nucleosome-cores when stabilized as Z-DNA through negative supercoiling. We have found that nucleosome assembly at the d(CG/GC)12 insert is prevented when the sequence is stable in the Z-conformation but it is not affected at all when the sequence adopts the right-handed B-form.     

Dependence of the linking deficiency of supercoiled minichromosomes upon nucleosome distortion.

The contribution from each nucleosome to the linking number of minichrosome DNA depends on two factors. These are the wrapping number, omega, which is the number of times the DNA wraps about the axis of the nucleosome; and the winding number, phi, which is the number of base pairs on the nucleosome divided by the helical repeat of the DNA. If the nucleosome is distorted with DNA surface contacts being preserved, phi remains unchanged. The wrapping number may still change, however, depending on the extent of the distortion. For example, if the usual cylindrical shape of the nucleosome is deformed into an ellipsoid while preserving the equatorial radius, then the wrapping number will increase. We apply these concepts to minichromosomes torsionally stressed by supercoiling with, for example, DNA gyrase. We analyze the experimental result that the maximum amount of supercoiling obtained by gyrase treatment of minichromosomes is the same as that of naked DNA. In particular, we show that this phenomenon can be explained by a relatively slight distortion of the nucleosome core while maintaining the surface contacts of the DNA on the core.     

Topoisomerase function during replication-independent chromatin assembly in yeast.

DNA topoisomerases I and II are the two major nuclear enzymes capable of relieving torsional strain in DNA. Of these enzymes, topoisomerase I plays the dominant role in relieving torsional strain during chromatin assembly in cell extracts from oocytes, eggs, and early embryos. We tested if the topoisomerases are used differentially during chromatin assembly in Saccharomyces cerevisiae by a combined biochemical and pharmacological approach. As measured by plasmid supercoiling, nucleosome deposition is severely impaired in assembly extracts from a yeast mutant with no topoisomerase I and a temperature-sensitive form of topoisomerase II (strain top1-top2). Expression of wild-type topoisomerase II in strain top1-top2 fully restored assembly-driven supercoiling, and assembly was equally efficient in extracts from strains expressing either topoisomerase I or II alone. Supercoiling in top1-top2 extract was rescued by adding back either purified topoisomerase I or II. Using the topoisomerase II poison VP-16, we show that topoisomerase II activity during chromatin assembly is the same in the presence and absence of topoisomerase I. We conclude that both topoisomerases I and II can provide the DNA relaxation activity required for efficient chromatin assembly in mitotically cycling yeast cells.     

Assembly of nucleosome-like structures mediated by cauliflower DNA topoisomerase

Nucleosome-like structures have been efficiently assembled in vitro by interaction of cauliflower histones, pBR322 DNA and cauliflower DNA topoisomerase, as assayed by supercoiling of relaxed circular DNA and by digestion with micrococcal nuclease. The optimum ionic strength for supercoiling was 150 mM KCl and the optimum weight ratio of histone to DNA was approximately 1.0. Four histones, H2A, H2B, H3 and H4, were necessary for the optimum assembling conditions, and the nucleosomes assembled protected DNA fragments of approximately 150 bp in length. It was found that cauliflower DNA topoisomerase acts not only as a DNA-relaxing enzyme but also as a chaperon factor for nucleosome assembly.     

Electrophoretic mobility of supercoiled,catenated and knotted DNA molecules

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.