The presence of two mitochondrial DNA topoisomerases in human acute leukemia cells

We have undertaken a study of DNA topoisomerases in mitochondria from human acute leukemia cells. Two activities have been detected in these organelles. One of the enzymes is presumably a type II topoisomerase, i.e., in ATP-dependent reactions it can catenate closed circular plasmid DNA, and decatenate closed circular kinetoplast DNA. A second topoisomerase is presumably a type I enzyme since, it can relax positive as well as negative supercoils in an ATP-independent reaction, it is unable to catenate plasmid DNA or decatenate kinetoplast DNA, and it is inhibited, rather than stimulated, by ATP.     

The purification and characterization of DNA topoisomerases I and II of the yeast Saccharomyces cerevisiae

The ATP-independent type I and the ATP-dependent type II DNA topoisomerase of the yeast Saccharomyces cerevisiae have been purified to near homogeneity, and the purification procedures are reported. Both purified topoisomerases are single subunit enzymes with monomer weights of Mr = 90,000 and 150,000 for the type I and type II enzyme, respectively. Sedimentation and gel filtration data suggest that the type I enzyme is monomeric and the type II enzyme is dimeric. Similar to other purified eukaryotic topoisomerases, the yeast type I enzyme does not require a divalent cation for activity, but is stimulated 10-20-fold in the presence of 7-10 mM Mg(II) or Ca(II). Mn(II) is about 25% as efficient as Mg(II) in this stimulation but Co(II) is inhibitory. The yeast type II topoisomerase has an absolute requirement for a divalent cation: Mg(II) is the most effective, whereas Mn(II), Ca(II), or Co(II) supports the reaction to a lesser extent. The type II enzyme also requires ATP or dATP; the nonhydrolyzable ATP analogues adenylyl imidodiphosphate and adenylyl (beta,gamma-methylene)diphosphonate are potent inhibitors. Both yeast topoisomerases are completely inhibited by N-ethylmaleimide at 0.5 mM. In addition, the type II enzyme, but not the type I enzyme, is inhibited to various extents by coumermycin, ethidium, and berenil. Both topoisomerases are nuclear enzymes; no topoisomerase specific to mitochondria has been detected.     

Structure of the topoisomerase VI-B subunit: implications for type II topoisomerase mechanism and evolution

Type IIA and type IIB topoisomerases each possess the ability to pass one DNA duplex through another in an ATP-dependent manner. The role of ATP in the strand passage reaction is poorly understood, particularly for the type IIB (topoisomerase VI) family. We have solved the structure of the ATP-binding subunit of topoisomerase VI (topoVI-B) in two states: an unliganded monomer and a nucleotide-bound dimer. We find that topoVI-B is highly structurally homologous to the entire 40-43 kDa ATPase region of type IIA topoisomerases and MutL proteins. Nucleotide binding to topoVI-B leads to dimerization of the protein and causes dramatic conformational changes within each protomer. Our data demonstrate that type IIA and type IIB topoisomerases have descended from a common ancestor and reveal how ATP turnover generates structural signals in the reactions of both type II topoisomerase families. When combined with the structure of the A subunit to create a picture of the intact topoisomerase VI holoenzyme, the ATP-driven motions of topoVI-B reveal a simple mechanism for strand passage by the type IIB topoisomerases.     

Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break

The T4 DNA topoisomerase is a recently discovered multisubunit protein that appears to have an essential role in the initiation of T4 bacteriophage DND replication. Treatment of double-stranded circular DNA with large amounts of this topoisomerase in the absence of ATP yields new DNA species which are knotted topological isomers of the double-stranded DNA circle. These knotted DNA circles, whether covalently closed or nicked, are converted to unknotted circles by treatment with trace amounts of the T4 topoisomerase in the presence of ATP. Very similar ATP-dependent enzyme activities capable of unknotting DNA are present in extracts of Drosophila eggs. Xenopus laevis eggs and mammalian tissue culture cells. The procaryotic enzyme, DNA gyrase, is also capable of unknotting DNA. We propose that these unknotting enzymes constitute a new general class of DNA topoisomerases (type II DNA topoisomerases). These enzymes must act via mechanisms that involve the concerted cleavage and rejoining of two opposite DNA strands, such that the DNA double helix is transiently broken. The passage of a second double-stranded DNA segment through this reversible double-strand break results in a variety of DNA topoisomerization reactions, including relaxation:super-coiling; knotting:unknotting and catenation:decatenation. In support of this type of mechanism, we demonstrate that the T4 DNA topoisomerase changes the linking number of a covalently closed double-stranded circular DNA molecule only by multiples of two. We discuss the possible roles of such enzymes in a variety of biological functions, along with their probable molecular mechanisms.     

DNA topoisomerases from rat liver: physiological variations

Besides the nicking-closing (topoisomerase I) activity, an ATP-dependent DNA topoisomerase is present in rat liver nuclei. The enzyme, partially purified, is able to catenate in vitro closed DNA circles in a magnesium-dependent, ATP-dependent, histone H1-dependent reaction, and to decatenate in vitro kinetoplast DNA networks to yield free minicircles in a magnesium-dependent and ATP-dependent reaction. It is largely similar to other eukaryotic type II topoisomerases in its requirements, and presumably belongs to this class of enzymes. Type I and type II activities were measured in rat liver nuclei as a function of regenerating time after partial hepatectomy: type I activity was not significantly changed during this process. In contrast, type II activity was considerably increased, suggesting a possible involvement of the enzyme in DNA replication.     

Adenosine 5'-O-(3-thio)triphosphate (ATPgammaS) promotes positive supercoiling of DNA by T. maritima reverse gyrase

Reverse gyrases are topoisomerases that catalyze ATP-dependent positive supercoiling of circular covalently closed DNA. They consist of an N-terminal helicase-like domain, fused to a C-terminal topoisomerase I-like domain. Most of our knowledge on reverse gyrase-mediated positive DNA supercoiling is based on studies of archaeal enzymes. To identify general and individual properties of reverse gyrases, we set out to characterize the reverse gyrase from a hyperthermophilic eubacterium. Thermotoga maritima reverse gyrase relaxes negatively supercoiled DNA in the presence of ADP or the non-hydrolyzable ATP-analog ADPNP. Nucleotide binding is necessary, but not sufficient for the relaxation reaction. In the presence of ATP, positive supercoils are introduced at temperatures above 50 degrees C. However, ATP hydrolysis is stimulated by DNA already at 37 degrees C, suggesting that reverse gyrase is not frozen at this temperature, but capable of undergoing inter-domain communication. Positive supercoiling by reverse gyrase is strictly coupled to ATP hydrolysis. At the physiological temperature of 75 degrees C, reverse gyrase binds and hydrolyzes ATPgammaS. Surprisingly, ATPgammaS hydrolysis is stimulated by DNA, and efficiently promotes positive DNA supercoiling, demonstrating that inter-domain communication during positive supercoiling is fully functional with both ATP and ATPgammaS. These findings support a model for communication between helicase-like and topoisomerase domains in reverse gyrase, in which an ATP and DNA-induced closure of the cleft in the helicase-like domain initiates a cycle of conformational changes that leads to positive DNA supercoiling.     

Differential modulation by spermidine of reactions catalyzed by type 1 prokaryotic and eukaryotic topoisomerases

In the absence of DNA aggregation, spermidine inhibited the relaxation of negatively supercoiled DNA by Escherichia coli topoisomerase I at concentrations of the polyamine normally found intracellularly. Spermidine also curtailed the cleavage of negatively supercoiled ColE1 DNA by the enzyme in the absence of Mg2+. On the contrary, knotting of M13 single-stranded DNA circles catalyzed by topoisomerase I was stimulated by the polyamine. Relaxation of supercoiled DNA by eukaryotic type 1 topoisomerases, such as calf thymus topoisomerase I and wheat germ topoisomerase, was significantly stimulated by spermidine in the same range of concentrations that inhibited the prokaryotic enzyme. In reactions catalyzed by S1 nuclease, the polyamine enhanced the digestion of single-stranded DNA and inhibited the nicking of negatively supercoiled DNA. These results suggest that spermidine modifies the supercoiled duplex substrate in these reactions by modulating the degree of single strandedness.     

Reverse gyrase, the two domains intimately cooperate to promote positive supercoiling

Reverse gyrases are atypical topoisomerases present in hyperthermophiles and are able to positively supercoil a circular DNA. Despite a number of studies, the mechanism by which they perform this peculiar activity is still unclear. Sequence data suggested that reverse gyrases are composed of two putative domains, a helicase-like and a topoisomerase I, usually in a single polypeptide. Based on these predictions, we have separately expressed the putative domains and the full-length polypeptide of Sulfolobus acidocaldarius reverse gyrase as recombinant proteins in Escherichia coli. We show the following. (i) The full-length recombinant enzyme sustains ATP-dependent positive supercoiling as efficiently as the wild type reverse gyrase. (ii) The topoisomerase domain exhibits a DNA relaxation activity by itself, although relatively low. (iii) We failed to detect helicase activity for both the N-terminal domain and the full-length reverse gyrase. (iv) Simple mixing of the two domains reconstitutes positive supercoiling activity at 75 degrees C. The cooperation between the domains seems specific, as the topoisomerase domain cannot be replaced by another thermophilic topoisomerase I, and the helicase-like cannot be replaced by a true helicase. (v) The helicase-like domain is not capable of promoting stoichiometric DNA unwinding by itself; like the supercoiling activity, unwinding requires the cooperation of both domains, either separately expressed or in a single polypeptide. However, unwinding occurs in the absence of ATP and DNA cleavage, indicating a structural effect upon binding to DNA. These results suggest that the N-terminal domain does not directly unwind DNA but acts more likely by driving ATP-dependent conformational changes within the whole enzyme, reminiscent of a protein motor.     

Yeast DNA topoisomerase II. An ATP-dependent type II topoisomerase that catalyzes the catenation, decatenation, unknotting, and relaxation of double-stranded DNA rings

An activity from the yeast Saccharomyces cerevisiae, initially noted for its catalysis of aggregation of covalently closed double-stranded DNA rings in the presence of ATP, has been identified as a type II DNA topoisomerase and is designated yeast DNA topoisomerase II. The formation of the DNA aggregate, which has been shown to be a network of DNA rings that are topologically interlocked, requires the presence of a yeast DNA-binding protein in addition to the topoisomerase. In the absence of the binding protein, yeast DNA topoisomerase II catalyzes decatenation and unknotting of duplex DNA rings and the relaxation of negatively or positively supercoiled DNA. All reactions are ATP-dependent and require Mg(II). Similar to other eukaryotic and phage T4-type II DNA topoisomerases, the yeast enzyme does not catalyze DNA supercoiling under the assay conditions employed. The activity is not sensitive to the gyrase inhibitor nalidixic acid, oxolinic acid, or novobiocin. Coumermycin inhibits the activity, however, at a concentration as low as 5 microgram/ml.     

Induction of mammalian DNA topoisomerase I and II mediated DNA cleavage by saintopin, a new antitumor agent from fungus.

Saintopin is an antitumor antibiotic recently discovered in mechanistically oriented screening using purified calf thymus DNA topoisomerases. Saintopin induced topoisomerase I mediated DNA cleavage comparable to that of camptothecin, and topoisomerase II mediated DNA cleavage equipotent to those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) or 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16). Treatment of a reaction mixture containing saintopin and topoisomerase I or II with either elevated temperature (65 degrees C) or higher salt concentration (0.5 M NaCl) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I and II mediated DNA cleavage induced by saintopin is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Consistent with the cleavable complex formation with both topoisomerases, saintopin inhibited catalytic activities of both topoisomerase I and topoisomerase II. The DNA cleavage intensity pattern induced by saintopin with topoisomerase I was different from that by camptothecin. A difference in cleavage pattern was also detected between saintopin and m-AMSA or VP-16 in topoisomerase II mediated DNA cleavage. DNA unwinding assay using T4 DNA ligase showed that saintopin is a weak DNA intercalator like m-AMSA. Thus, saintopin represents a new class of antitumor agent that can induce both mammalian DNA topoisomerase I and mammalian DNA topisomerase II mediated DNA cleavage.     

Reverse gyrase; ATP-dependent type I topoisomerase from Sulfolobus

Reverse gyrase, a topoisomerase which introduces positive superhelical turns into DNA, has been purified from Sulfolobus to near homogeneity. It is a single polypeptide with a mol. wt. of 120 000 as determined by denaturing gel electrophoresis. Contrary to a previous report, it is a type I topoisomerase as judged by the linking-number change of closed circular DNA topoisomer. Unlike other known type I topoisomerases, ATP or dATP is required for introducing positive superhelical turns. In order to relax negatively supercoiled DNA, other nucleotide triphosphates (XTP) are also effective with low efficiency. In the absence of either XTP or divalent cations, the enzyme introduces nicks into closed circular DNA when the reaction is stopped by SDS. This suggests that reverse gyrase cuts one of the two strands of DNA in the course of its enzymatic reaction.     

DNA supercoiling and relaxation by ATP-dependent DNA topoisomerases.

Bacterial DNA gyrase and the eukaryotic type II DNA topoisomerases are ATPases that catalyse the introduction or removal of DNA supercoils and the formation and resolution of DNA knots and catenanes. Gyrase is unique in using ATP to drive the energetically unfavourable negative supercoiling of DNA, an example of mechanochemical coupling: in contrast, eukaryotic topoisomerase II relaxes DNA in an ATP-requiring reaction. In each case, the enzyme-DNA complex acts as a 'gate' mediating the passage of a DNA segment through a transient enzyme-bridged double-strand DNA break. We are using a variety of genetic and enzymic approaches to probe the nature of these complexes and their mechanism of action. Recent studies will be described focusing on the role of DNA wrapping on the A2B2 gyrase complex, subunit activities uncovered by using ATP analogues and the coumarin and quinolone inhibitors, and the identification and functions of discrete subunit domains. Homology between gyrase subunits and the A2 homodimer of eukaryotic topo II suggests functional conservation between these proteins. The role of ATP hydrolysis by these topoisomerases will be discussed in regard to other energy coupling systems.     

DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI

Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by type I restriction enzymes do not appear to be the trigger for DNA cleavage.     

Catenation of DNA by eucaryotic topoisomerase II associated with simian virus 40 minichromosomes

After incubation of purified SV40 minichromosomes with superhelical DNA molecules either of SV40 or plasmid origin, a catenation of monomeric DNA via dimers and multimers to large networks was observed. The catenation reaction was stimulated by the DNA condensing agent spermidine with ATP as an energy donor and was dependent on the presence of magnesium ions. The reaction could be blocked by inhibitors of topoisomerase II such as novobiocin and nalidixic acid. Relaxed covalently closed circular DNA was catenated to networks in the presence of ATP as the energy donor.     

Direct measurement of DNA bending by type IIA topoisomerases: implications for non-equilibrium topology simplification

Type IIA topoisomerases modify DNA topology by passing one segment of duplex DNA (transfer or T–segment) through a transient double-strand break in a second segment of DNA (gate or G–segment) in an ATP-dependent reaction. Type IIA topoisomerases decatenate, unknot and relax supercoiled DNA to levels below equilibrium, resulting in global topology simplification. The mechanism underlying this non-equilibrium topology simplification remains speculative. The bend angle model postulates that non-equilibrium topology simplification scales with the bend angle imposed on the G–segment DNA by the binding of a type IIA topoisomerase. To test this bend angle model, we used atomic force microscopy and single-molecule Förster resonance energy transfer to measure the extent of bending imposed on DNA by three type IIA topoisomerases that span the range of topology simplification activity. We found that Escherichia coli topoisomerase IV, yeast topoisomerase II and human topoisomerase IIα each bend DNA to a similar degree. These data suggest that DNA bending is not the sole determinant of non-equilibrium topology simplification. Rather, they suggest a fundamental and conserved role for DNA bending in the enzymatic cycle of type IIA topoisomerases.     

The mechanism of negative DNA supercoiling: A cascade of DNA-induced conformational changes prepares gyrase for strand passage

DNA topoisomerases inter-convert different DNA topoisomers in the cell. They catalyze the introduction or relaxation of DNA supercoils, as well as catenation and decatenation. Members of the type I topoisomerase family cleave a single strand of their double-stranded DNA substrate, whereas enzymes of the type II family cleave both DNA strands. Bacterial DNA gyrase, a type II topoisomerase, catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. Gyrase is not present in humans, and constitutes an attractive drug target for the treatment of bacterial and parasite infections. DNA supercoiling by gyrase is believed to occur by a strand passage mechanism, in which one segment of the double-stranded DNA substrate is passed through a (transient) break in a second segment. This mechanism requires the coordinated opening and closing of three protein interfaces, so-called gates, to ensure the directionality of strand passage toward negative supercoiling.Single molecule fluorescence resonance energy transfer experiments are ideally suited to investigate conformational changes during the catalytic cycle of DNA topoisomerases. In this review, we summarize the current knowledge on the cascade of DNA- and nucleotide-induced conformational changes in gyrase that lead to strand passage and negative supercoiling of DNA. We discuss how these conformational changes couple ATP hydrolysis to DNA supercoiling in gyrase, and how the common mechanistic principle of coordinated gate opening and closing is modulated to allow for the catalysis of different reactions by different type II topoisomerases.     

Intrinsic DNA-dependent ATPase activity of reverse gyrase

Reverse gyrase is a type I DNA topoisomerase that promotes positive supercoiling of closed-circular double-stranded DNA through an ATP-dependent reaction, and it was purified from an archaebacterium, Sulfolobus. When ATP is replaced by UTP, GTP, or CTP, this enzyme just relaxes the negatively supercoiled closed-circular double-stranded DNA. We found that reverse gyrase hydrolyzes ATP through a double-stranded DNA-dependent reaction. The superhelicity of the DNA did not affect the ATPase activity. However, reverse gyrase does not hydrolyze UTP, GTP, or CTP. Therefore, any of the four nucleotide 5'-triphosphates acts as an effector for the topoisomerase activity of reverse gyrase, but only ATP supports the positive supercoiling of closed-circular double-stranded DNA, through the energy released on its hydrolysis. Single-stranded DNA was a much more potent cofactor for the ATPase activity of the enzyme than double-stranded DNA, and it acted as a potent inhibitor for the topoisomerase activity on double-stranded DNA. These results indicate that reverse gyrase has higher affinity to single-stranded DNA than to double-stranded DNA, which suggests a cellular function of the enzyme.     

Reprint of “The mechanism of negative DNA supercoiling: A cascade of DNA-induced conformational changes prepares gyrase for strand passage”

DNA topoisomerases inter-convert different DNA topoisomers in the cell. They catalyze the introduction or relaxation of DNA supercoils, as well as catenation and decatenation. Members of the type I topoisomerase family cleave a single strand of their double-stranded DNA substrate, whereas enzymes of the type II family cleave both DNA strands. Bacterial DNA gyrase, a type II topoisomerase, catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. Gyrase is not present in humans, and constitutes an attractive drug target for the treatment of bacterial and parasite infections. DNA supercoiling by gyrase is believed to occur by a strand passage mechanism, in which one segment of the double-stranded DNA substrate is passed through a (transient) break in a second segment. This mechanism requires the coordinated opening and closing of three protein interfaces, so-called gates, to ensure the directionality of strand passage toward negative supercoiling.Single molecule fluorescence resonance energy transfer experiments are ideally suited to investigate conformational changes during the catalytic cycle of DNA topoisomerases. In this review, we summarize the current knowledge on the cascade of DNA- and nucleotide-induced conformational changes in gyrase that lead to strand passage and negative supercoiling of DNA. We discuss how these conformational changes couple ATP hydrolysis to DNA supercoiling in gyrase, and how the common mechanistic principle of coordinated gate opening and closing is modulated to allow for the catalysis of different reactions by different type II topoisomerases.     

How Do Type II Topoisomerases Use ATP Hydrolysis to Simplify DNA Topology beyond Equilibrium? Investigating the Relaxation Reaction of Nonsupercoiling Type II Topoisomerases

DNA topoisomerases control the topology of DNA (e.g., the level of supercoiling) in all cells. Type IIA topoisomerases are ATP-dependent enzymes that have been shown to simplify the topology of their DNA substrates to a level beyond that expected at equilibrium (i.e., more relaxed than the product of relaxation by ATP-independent enzymes, such as type I topoisomerases, or a lower-than-equilibrium level of catenation). The mechanism of this effect is currently unknown, although several models have been suggested. We have analyzed the DNA relaxation reactions of type II topoisomerases to further explore this phenomenon. We find that all type IIA topoisomerases tested exhibit the effect to a similar degree and that it is not dependent on the supercoil-sensing C-terminal domains of the enzymes. As recently reported, the type IIB topoisomerase, topoisomerase VI (which is only distantly related to type IIA enzymes), does not exhibit topology simplification. We find that topology simplification is not significantly dependent on circle size in the range ∼ 2-9 kbp and is not altered by reducing the free energy available from ATP hydrolysis by varying the ADP:ATP ratio. A direct test of one model (DNA tracking; i.e., sliding of a protein clamp along DNA to trap supercoils) suggests that this is unlikely to be the explanation for the effect. We conclude that geometric selection of DNA segments by the enzymes is likely to be a primary source of the effect, but that it is possible that other kinetic factors contribute. We also speculate whether topology simplification might simply be an evolutionary relic, with no adaptive significance.     

ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings

Extracts of Drosophila embryos contain an enzymatic activity that converts circular DNAs into huge networks of catenated rings in an ATP-dependent fashion. The catenation activity is resolved into two protein components during purification. One component is a novel DNA topoisomerase that requires the presence of ATP in order to relax supercoiled DNA. We have shown that the ATP-dependent DNA topoisomerase relaxes DNA by a mechanism distinct from that of nicking-closing enzymes. The Drosophila ATP-dependent topoisomerase allows one segment of a circular DNA to pass through transient breaks in both strands at another site on the DNA circle without any relative rotation between the ends at the transient break. This mechanism can convert negative supertwists to positive twists and vice versa until a relaxed equilibrium state is reached. The formation of catenated rings is mediated by an analogous bimolecular reaction which can occur between two nonhomologous DNA circles. The catenation reaction is fully reversible: in the presence of the second protein component, circular DNA is converted quantitatively into catenated forms; in its absence, the ATP-dependent topoisomerase resolves catenated networks back into monomer circles. The Drosophila ATP-dependent topoisomerase appears to be closely related to E. coli DNA gyrase in that both use a similar mechanism to change the topology of DNA, both require ATP and both are inhibited by the antibiotic novobiocin. The presence of an enzyme that allows one DNA helix to pass freely through another could not only be useful in relaxation of topological constraints, but also may be involved in the folding and unfolding of eucaryotic chromosomes.