Nucleosome Assembly Depends on the Torsion in the DNA Molecule: A Magnetic Tweezers Study

We have used magnetic tweezers to study nucleosome assembly on topologically constrained DNA molecules. Assembly was achieved using chicken erythrocyte core histones and histone chaperone protein Nap1 under constant low force. We have observed only partial assembly when the DNA was topologically constrained and much more complete assembly on unconstrained (nicked) DNA tethers. To verify our hypothesis that the lack of full nucleosome assembly on topologically constrained tethers was due to compensatory accumulation of positive supercoiling in the rest of the template, we carried out experiments in which we mechanically relieved the positive supercoiling by rotating the external magnetic field at certain time points of the assembly process. Indeed, such rotation did lead to the same nucleosome saturation level as in the case of nicked tethers. We conclude that levels of positive supercoiling in the range of 0.025-0.051 (most probably in the form of twist) stall the nucleosome assembly process.     

Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs.

A cell-free system from Xenopus eggs mimics the reaction occurring at the eukaryotic replicative fork in vivo when chromatin assembly is coupled to complementary strand synthesis of DNA. DNA synthesis on single-stranded circular DNA promotes supercoiling and the replicated molecule sediments as a discrete nucleoprotein complex. Micrococcal nuclease digestion reveals a characteristic pattern of multiples of 200 bp of DNA. The kinetics of chromatin assembly and DNA synthesis are coincident and both processes occur with a rate comparable with chromosomal replication in vivo in early embryos. The DNA synthesis reaction can be uncoupled from the assembly reaction. Thus, titration of chromatin proteins by preincubation of the extract with double-stranded DNA prevents the supercoiling of replicated DNA without affecting the rate of synthesis. In contrast, chromatin assembly performed on unreplicated double-stranded DNA is a slower process as compared with the assembly coupled to DNA synthesis. Supercoiled molecules are detected after 30 min replication whereas at least 2 h are required to observe the first form I DNA with unreplicated double-stranded DNA. Such a system where chromatin assembly is promoted by DNA synthesis should be valuable for studying the interaction of specific factors with DNA during chromatin assembly at the replicative fork.     

Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.

We describe a dominant behavioral marker, rol-6(su-1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double-strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single-stranded oligonucleotide was co-injected with the double-stranded DNA.     

Homogeneous repair of nuclear genes after experimental stroke

The repair of oxidative DNA lesions (ODLs) in the nucleus of ischemic cortical brain cells was examined following experimentally induced stroke by occluding the right middle cerebral artery and both common carotid arteries for 60-90 min followed by reperfusion in male long-Evans hooded rats. The control group consisted of sham-operated animals undergoing the same surgery without vessel occlusion. Using a gene-specific assay based upon the presence of Escherichia coli Fpg protein-sensitive sites, we noted that animals with stroke exhibited six and four ODLs per gene in the actin and DNA polymerase-beta genes, respectively. This was increased from one per four copies of each gene in the sham-operated control (p < 0.01). One half of the initial ODLs was repaired within 30 min, and 83% of them were repaired as early as 45 min of reperfusion. There was no further increase when gene repair was measured again at 2 h of reperfusion. The rates of active repair within 45 min of reperfusion were the same in these two genes (p = 0.103, ANOVA). BrdU (10 mg/kg) was administered via intraperitoneal injection at least one day before surgery. We observed that there was no significant incorporation of BrdU triphosphates into genomic DNA during active repair, but there were significant amounts of BrdU triphosphate in nuclear DNA after active repair. The result indicates that genomic repair of ODLs in the brain did not significantly incorporate BrdU, and the initiation of neurogenesis probably starts after the completion of repair in the brain.     

The ubiquitous chromatin protein DEK alters the structure of DNA by introducing positive supercoils

We have investigated the molecular mechanism by which the proto-oncogene protein DEK, an abundant chromatin-associated protein, changes the topology of DNA in chromatin in vitro. Band-shift assays and electron microscopy revealed that DEK induces both intra- and intermolecular interactions between DNA molecules. Binding of the DEK protein introduces constrained positive supercoils both into protein-free DNA and into DNA in chromatin. The induced change in topology is reversible after removal of the DEK protein. As shown by sedimentation analysis and electron microscopy, the DEK-induced positive supercoiling causes distinct structural changes of DNA and chromatin. The observed direct effects of DEK on chromatin folding help to understand the function that this major chromatin protein performs in the nucleus.     

Interplay of DNA supercoiling and catenation during the segregation of sister duplexes

The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo.     

Normal reconstruction of DNA supercoiling and chromatin structure in cockayne syndrome cells during repair of damage from ultraviolet light.

The chromatin of human cells undergoes structural rearrangements during excision repair of ultraviolet damage in DNA that were detected by transient relaxation of DNA supercoiling and increased staphylococcal nuclease digestibility of repaired sites. Inhibition of polymerization and/or ligation of repaired regions with inhibitors of DNA polymerase alpha (cytosine arabinoside and aphidicolin) resulted in the accumulation of single-strand breaks, delayed reconstruction of DNA supercoiling, and maintenance of the staphylococcal nuclease digestibility. These observations suggest that reconstruction of the native chromatin state requires completion of repaired regions with covalent ligation into the DNA strands. Although previous claims have been made that a late stage associated with ligation of repaired regions may be defective in cells from patients with Cockayne syndrome, complete reconstruction of the native chromatin occurred in cells from three unrelated patients after ultraviolet irradiation. No abnormality in repair was therefore detected in Cockayne syndrome cells. The hypersensitivity of cell survival and semiconservative DNA replication to damage by ultraviolet light in this human disorder must therefore be regarded as features of a primary defect in DNA metabolism unrelated to DNA repair.     

Intramuscular plasmid DNA electrotransfer: biodistribution and degradation

We have studied radiolabelled plasmid DNA biodistribution and degradation in the muscle at different times after injection, with or without electrotransfer using previously defined conditions. Radiolabelled plasmid progressively left the muscle and was degraded as soon as 5 min after plasmid injection, with or without electrotransfer. Autoradiography showed that the major part of injected radioactivity was detected in the interfibrilar space of a large proportion of the muscle. Large zones of accumulation of radioactivity, which seems to be contained in some fibres (more than 20 microm), were identified as soon as 5 min after electrotransfer. Such structures were never observed on slices of non-electrotransferred muscles. However, these structures were not frequent and probably lesional. The surprising fact is that despite the amount of intact plasmid having been greatly reduced between 5 min and 3 h after injection, the level of transfection remains unchanged whether electric pulses were delivered 20 s or 3 h after injection. Such a behavior was similarly observed when injecting 0.3, 3 or 30 microg of plasmid DNA. Moreover, the transfection level was correlated to the amount of plasmid DNA injected. These results suggest that as soon as it is injected, plasmid DNA is proportionally partitioned between at least two compartments. While a major part of plasmid DNA is rapidly cleared and degraded, the electrotransferable pool of plasmid DNA represents a very small part of the amount injected and belongs to another compartment where it is protected from endogenous DNAses.     

Supercoiling-dependent site-specific binding of HU to naked Mu DNA.

Using HU chemical nucleases to probe HU-DNA interactions, we report here for the first time site-specific binding of HU to naked DNA. An unique feature of this interaction is the absolute requirement for negative DNA supercoiling for detectable levels of site-specific DNA binding. The HU binding site is the Mu spacer between the L1 and L2 transposase binding sites. Our results suggest recognition of an altered DNA structure which is induced by DNA supercoiling. We propose that recruitment of HU to this naked DNA site induces the DNA bending required for productive synapsis and transpososome assembly. Implications of HU as a supercoiling sensor with a potential in vivo regulatory role are discussed. Finally, using HU nucleases we have also shown that non-specific DNA binding by HU is stimulated by increasing levels of supercoiling.     

Degradation of linear DNA by a strand-specific exonuclease activity in Xenopus laevis oocytes.

Linear DNA injected into Xenopus laevis oocyte nuclei recombines with high efficiency if homologous sequences are present at overlapping molecular ends. We found that injected linear DNA was degraded by a 5'----3' strand-specific exonuclease activity during incubation in the oocyte nucleus to leave a heterogeneous population of 3'-tailed molecules. Decreasing the concentration of DNA injected increased the heterogeneity and the average rate of degradation. The 3' tails created were relatively stable; among molecules persisting after overnight incubation, many had 3' tails intact to within 10 bases of the original ends. DNA molecules that were efficient substrates for homologous recombination in oocytes were also partially degraded, leaving 3' tails. We found no evidence for other potent nuclease activities. If molecules with recessed 3'-OH ends were injected, endogenous polymerase efficiently resynthesized complementary strands before degradation of the 5' tails occurred. 3'-tailed molecules are plausible intermediates in the initiation of homologous recombination events in Xenopus oocyte nuclei.     

Conformational and thermodynamic properties of supercoiled DNA.

We used Monte Carlo simulations to investigate the conformational and thermodynamic properties of DNA molecules with physiological levels of supercoiling. Three parameters determine the properties of DNA in this model: Kuhn statistical length, torsional rigidity and effective double-helix diameter. The chains in the simulation resemble strongly those observed by electron microscopy and have the conformation of an interwound superhelix whose axis is often branched. We compared the geometry of simulated chains with that determined experimentally by electron microscopy and by topological methods. We found a very close agreement between the Monte Carlo and experimental values for writhe, superhelix axis length and the number of superhelical turns. The computed number of superhelix branches was found to be dependent on superhelix density, DNA chain length and double-helix diameter. We investigated the thermodynamics of supercoiling and found that at low superhelix density the entropic contribution to superhelix free energy is negligible, whereas at high superhelix density, the entropic and enthalpic contributions are nearly equal. We calculated the effect of supercoiling on the spatial distribution of DNA segments. The probability that a pair of DNA sites separated along the chain contour by at least 50 nm are juxtaposed is about two orders of magnitude greater in supercoiled DNA than in relaxed DNA. This increase in the effective local concentration of DNA is not strongly dependent on the contour separation between the sites. We discuss the implications of this enhancement of site juxtaposition by supercoiling in the context of protein-DNA interactions involving multiple DNA-binding sites.     

Solution structures of DNA-bound gyrase

The DNA gyrase negative supercoiling mechanism involves the assembly of a large gyrase/DNA complex and conformational rearrangements coupled to ATP hydrolysis. To establish the complex arrangement that directs the reaction towards negative supercoiling, bacterial gyrase complexes bound to 137- or 217-bp DNA fragments representing the starting conformational state of the catalytic cycle were characterized by sedimentation velocity and small-angle X-ray scattering (SAXS) experiments. The experiments revealed elongated complexes with hydrodynamic radii of 70–80 Å. Molecular envelopes calculated from these SAXS data show 2-fold symmetric molecules with the C-terminal domain (CTD) of the A subunit and the ATPase domain of the B subunit at opposite ends of the complexes. The proposed gyrase model, with the DNA binding along the sides of the molecule and wrapping around the CTDs located near the exit gate of the protein, adds new information on the mechanism of DNA negative supercoiling.     

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.     

The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange

Histone chaperone Asf1 participates in heterochromatin silencing, DNA repair and regulation of gene expression, and promotes the assembly of DNA into chromatin in vitro. To determine the influence of Asf1 on genetic stability, we have analysed the effect of asf1Delta on homologous recombination. In accordance with a defect in nucleosome assembly, asf1Delta leads to a loss of negative supercoiling in plasmids. Importantly, asf1Delta increases spontaneous recombination between inverted DNA sequences. This increase correlates with an accumulation of double-strand breaks (DSBs) as determined by immunodetection of phosphorylated histone H2A and fluorescent detection of Rad52-YFP foci during S and G2/M phases. In addition, asf1Delta shows high levels of sister chromatid exchange (SCE) and is proficient in DSB-induced SCE as determined by physical analysis. Our results suggest that defective chromatin assembly caused by asf1Delta leads to DSBs that can be repaired by SCE, affecting genetic stability.     

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.