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PubMed1997

Approaches to determine stoichiometry of viral assembly components.

Due to the rapidity of biological reactions, it is difficult to isolate intermediates or to determine the stoichiometry of participants in intermediate reactions. Instead of determining the absolute amount of each component, this study involved the use of relative parameters, such as dilution factors, percentages probabilities, and slopes of titration curves, that can be more accurately quantified to determine the stoichiometry of components involved in bacteriophage phi29 assembly. This work takes advantage of the sensitive in vitro phage phi29 assembly system, in which 10(8) infectious virions per ml without background can be assembled from eight purified components. It provides a convenient assay for quantification of the stoichiometry of packaging components, including the viral procapsid, genomic DNA, DNA-packaging pRNA, and other structural proteins and enzymes. The presence of a procapsid binding domain and another essential functional domain within the pRNA makes it an ideal component for constructing lethal mutants for competitive procapsid binding. Two methods were used for stoichiometry determination. Method 1 was to determine the combination probability of mutant and wild-type pRNAs bound to procapsids. The probability of procapsids that possess a certain amount of mutant and a certain amount of wild-type pRNA, both with an equal binding affinity, was predicted with the binomial equation [EQUATION IN TEXT] where Z is the total number of pRNAs per procapsid, M is the number of mutant pRNAs bound to one procapsid, and (ZM) is equal to [FORMULA IN TEXT]. With various ratios of mutant to wild-type pRNA in in vitro viral assembly, the percent mutant pRNA versus the yield of virions was plotted and compared to a series of predicted curves to find a best fit. It was determined that five or six copies of pRNA were required for one DNA-packaging event, while only one mutant pRNA per procapsid was sufficient to block packaging. Method 2 involved the comparison of slopes of curves of dilution factors versus the yield of virions. Components with known stoichiometries served as standard controls. The larger the stoichiometry of the component, the more dramatic the influence of the dilution factor on the reaction. A slope of 1 indicates that one copy of the component is involved in the assembly of one virion. A slope larger than 1 would indicate multiple-copy involvement. By this method, the stoichiometry of gp11 in phi29 particles was determined to be approximately 12. These approaches are useful for the determination of the stoichiometry of functional units involved in viral assembly, be they single molecules or oligomers. However, these approaches are not suitable for the determination of exact copy numbers of individual molecules involved if the functional unit is composed of multiple subunits prior to assembly.

Bacillus Phages↗
PubMed1999

In vitro assembly properties of human immunodeficiency virus type 1 Gag protein lacking the p6 domain.

Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Deltap6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Deltap6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Deltap6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Deltap6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Deltap6 and nucleic acid are not sufficient for the formation of normal-sized particles.

Escherichia coli↗
PubMed2000

The simian virus 40 core origin contains two separate sequence modules that support T-antigen double-hexamer assembly.

Using subfragments of the simian virus 40 (SV40) core origin, we demonstrate that two alternative modules exist for the assembly of T-antigen (T-ag) double hexamers. Pentanucleotides 1 and 3 and the early palindrome (EP) constitute one assembly unit, while pentanucleotides 2 and 4 and the AT-rich region constitute a second, relatively weak, assembly unit. Related studies indicate that on the unit made up of pentanucleotide 1 and 3 and the EP assembly unit, the first hexamer forms on pentanucleotide 1 and that owing to additional protein-DNA and protein-protein interactions, the second hexamer is able to form on pentanucleotide 3. Oligomerization on the unit made up of pentanucleotide 2 and 4 and the AT-rich region is initiated by assembly of a hexamer on pentanucleotide 4; subsequent formation of the second hexamer takes place on pentanucleotide 2. Given that oligomerization on the SV40 origin is limited to double-hexamer formation, it is likely that only a single module is used for the initial assembly of T-ag double hexamers. Finally, we discuss the evidence that nucleotide hydrolysis is required for the remodeling events that result in the utilization of the second assembly unit.

Adenosine Triphosphate↗
PubMed2000

Role of the Rous sarcoma virus p10 domain in shape determination of gag virus-like particles assembled in vitro and within Escherichia coli.

Purified retrovirus Gag proteins can assemble in vitro into virus-like particles (VLPs) in the presence of RNA. It was shown previously that a Rous sarcoma virus Gag protein missing only the protease domain forms spherical particles resembling immature virions lacking a membrane but that a similar protein missing the p10 domain forms tubular particles. Thus, p10 plays a role in spherical particle formation. To further study this shape-determining function, we dissected the p10 domain by mutagenesis and examined VLPs assembled within Escherichia coli or assembled in vitro from purified proteins. The results identified a minimal contiguous segment of 25 amino acid residues at the C terminus of p10 that is sufficient to restore efficient spherical assembly to a p10 deletion mutant. Random and site-directed mutations were introduced into this segment of polypeptide, and the shapes of particles formed in E. coli were examined in crude extracts by electron microscopy. Three phenotypes were observed: tubular morphology, spherical morphology, or no regular structure. While the particle morphology visualized in crude extracts generally was the same as that visualized for purified proteins, some tubular mutants scored as spherical when tested as purified proteins, suggesting that a cellular factor may also play a role in shape determination. We also examined the assembly properties of smaller Gag proteins consisting of the capsid protein-nucleocapsid protein (CA-NC) domains with short N-terminal extensions or deletions. Addition of one or three residues allowed CA-NC to form spheres instead of tubes in vitro, but the efficiency of assembly was extremely low. Deletion of the N-terminal residue(s) abrogated assembly. Taken together, these results imply that the N terminus of CA and the adjacent upstream 25 residues play an important role in the polymerization of the Gag protein.

Amino Acid Sequence↗
PubMed2000

The carboxy-terminal fragment of nucleolin interacts with the nucleocapsid domain of retroviral gag proteins and inhibits virion assembly.

A yeast two-hybrid screen for cellular proteins that interact with the murine leukemia virus (MuLV) Gag protein resulted in the identification of nucleolin, a host protein known to function in ribosome assembly. The interacting fusions contained the carboxy-terminal 212 amino acids of nucleolin [Nuc(212)]. The nucleocapsid (NC) portion of Gag was necessary and sufficient to mediate the binding to Nuc(212). The interaction of Gag with Nuc(212) could be demonstrated in vitro and was manifested in vivo by the NC-dependent incorporation of Nuc(212) inside MuLV virions. Overexpression of Nuc(212), but not full-length nucleolin, potently and specifically blocked MuLV virion assembly and/or release. A mutant of MuLV, selected to specifically disrupt the binding to Nuc(212), was found to be severely defective for virion assembly. This mutant harbors a single point mutation in capsid (CA) adjacent to the CA-NC junction, suggesting a role for this region in Moloney MuLV assembly. These experiments demonstrate that selection for proteins that bind assembly domain(s) can yield potent inhibitors of virion assembly. These experiments also raise the possibility that a nucleolin-Gag interaction may be involved in virion assembly.

Animals↗
PubMed2001

In vitro assembly of Sindbis virus core-like particles from cross-linked dimers of truncated and mutant capsid proteins.

A nucleic acid-bound capsid protein dimer was previously identified using a Sindbis virus in vitro nucleocapsid assembly system and cross-linking reagents. Cross-link mapping, in combination with a model of the nucleocapsid core, suggested that this dimer contained one monomer from each of two adjacent capsomeres. This intercapsomere dimer is believed to be the initial intermediate in the nucleocapsid core assembly mechanism. This paper presents the purification of cross-linked dimers of a truncated capsid protein and the partial purification of cross-linked dimers of a full-length assembly-defective mutant. The assembly of core-like particles from these cross-linked capsid protein dimers is demonstrated. Core-like particles generated from cross-linked full-length mutant CP(19-264)L52D were examined by electron microscopy and appeared to have a morphology similar to that of wild-type in vitro-assembled core-like particles, although a slight size difference was often visible. Truncated cross-linked CP(81-264) dimers generated core-like particles as well. These core-like particles could subsequently be disassembled when reversible cross-linking reagents were used to form the dimers. The ability of the covalent intercapsomere cross-link to rescue capsid proteins with assembly defects or truncations in the amino-terminal region of the capsid protein supports the previous model of assembly and suggests a possible role for the amino-terminal region of the protein.

Blotting, Western↗
PubMed2001

Mouse-human heterokaryons support efficient human immunodeficiency virus type 1 assembly.

Murine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication. Mus musculus lymphoid cell lines EL4 and L1-2 and Mus dunni fibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infected M. dunni tail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.

3T3 Cells↗
PubMed2004

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.

Amino Acid Sequence↗
PubMed2005

Herpes simplex virus tegument protein VP22 contains an internal VP16 interaction domain and a C-terminal domain that are both required for VP22 assembly into the virus particle.

Many steps along the herpesvirus assembly and maturation pathway remain unclear. In particular, the acquisition of the virus tegument is a poorly understood process, and the molecular interactions involved in tegument assembly have not yet been defined. Previously we have shown that the two major herpes simplex virus tegument proteins VP22 and VP16 are able to interact, although the relevance of this to virus assembly is not clear. Here we have constructed a number of recombinant viruses expressing N- and C-terminal truncations of VP22 and have used them to identify regions of the protein involved in its assembly into the virus structure. Analysis of the packaging of these VP22 variants into extracellular virions revealed that the C terminus of VP22 is absolutely required for this process, with removal of the C-terminal 89 residues abrogating its incorporation. However, while these 89 residues alone were sufficient for specific incorporation of small amounts of VP22 into the tegument, efficient packaging of VP22 to the levels of full-length protein required an additional 52 residues of the protein. Coimmunoprecipitation assays indicated that these 52 residues also contained the interaction domain for VP16. Furthermore, analysis of the subcellular localization of the mutant forms of VP22 revealed that only those truncations that were efficiently assembled formed characteristic cytoplasmic trafficking complexes, suggesting that these complexes may represent the cellular location for VP22 assembly into the virus. Taken together, these results suggest that there are two determinants involved in the packaging of VP22-a C-terminal domain and an internal VP16 interaction domain, both of which are required for the efficient recruitment of VP22 to sites of virus assembly.

Animals↗
PubMed2006

Dynamic pathways for viral capsid assembly.

We develop a class of models with which we simulate the assembly of particles into T1 capsidlike objects using Newtonian dynamics. By simulating assembly for many different values of system parameters, we vary the forces that drive assembly. For some ranges of parameters, assembly is facile; for others, assembly is dynamically frustrated by kinetic traps corresponding to malformed or incompletely formed capsids. Our simulations sample many independent trajectories at various capsomer concentrations, allowing for statistically meaningful conclusions. Depending on subunit (i.e., capsomer) geometries, successful assembly proceeds by several mechanisms involving binding of intermediates of various sizes. We discuss the relationship between these mechanisms and experimental evaluations of capsid assembly processes.

Capsid↗
PubMed1999

A role for the clathrin assembly domain of AP180 in synaptic vesicle endocytosis.

We have used the squid giant synapse to determine whether clathrin assembly by AP180 is important for synaptic vesicle endocytosis. The squid homolog of AP180 encodes a 751 amino acid protein with 40% sequence identity to mouse AP180. Alignment of squid AP180 with other AP180 homologs shows that amino acid identity was highest in the N-terminal inositide-binding domain of the protein and weakest in the C-terminal clathrin assembly domain. Recombinant squid AP180 was able to assemble clathrin in vitro, suggesting a conserved three-dimensional structure that mediates clathrin assembly despite the divergent primary sequence of the C-terminal domain. Microinjection of the C-terminal domains of either mouse or squid AP180 into the giant presynaptic terminal of squid enhanced synaptic transmission. Conversely, a peptide from the C-terminal domain of squid AP180 that inhibited clathrin assembly in vitro completely blocked synaptic transmission when it was injected into the giant presynaptic terminal. This inhibitory effect occurred over a time scale of minutes when the synapse was stimulated at low (0.03 Hz), physiological rates. Electron microscopic analysis revealed several structural changes consistent with the inhibition of synaptic vesicle endocytosis; peptide-injected terminals had far fewer synaptic vesicles, were depleted of coated vesicles, and had a larger plasma membrane perimeter than terminals injected with control solutions. In addition, the remaining synaptic vesicles were significantly larger in diameter. We conclude that the clathrin assembly domain of AP180 is important for synaptic vesicle recycling at physiological rates of activity and that assembly of clathrin by AP180 is necessary for maintaining a pool of releasable synaptic vesicles.

Adaptor Proteins, Vesicular Transport↗
PubMed2000

A conserved clathrin assembly motif essential for synaptic vesicle endocytosis.

Although clathrin assembly by adaptor proteins (APs) plays a major role in the recycling of synaptic vesicles, the molecular mechanism that allows APs to assemble clathrin is poorly understood. Here we demonstrate that AP180, like AP-2 and AP-3, binds to the N-terminal domain of clathrin. Sequence analysis reveals a motif, containing the sequence DLL, that exists in multiple copies in many clathrin APs. Progressive deletion of these motifs caused a gradual reduction in the ability of AP180 to assemble clathrin in vitro. Peptides from AP180 or AP-2 containing this motif also competitively inhibited clathrin assembly by either protein. Microinjection of these peptides into squid giant presynaptic terminals reversibly blocked synaptic transmission and inhibited synaptic vesicle endocytosis by preventing coated pit formation at the plasma membrane. These results indicate that the DLL motif confers clathrin assembly properties to AP180 and AP-2 and, perhaps, to other APs. We propose that APs promote clathrin assembly by cross-linking clathrin triskelia via multivalent interactions between repeated DLL motifs in the APs and complementary binding sites on the N-terminal domain of clathrin. These results reveal the structural basis for clathrin assembly and provide novel insights into the molecular mechanism of clathrin-mediated synaptic vesicle endocytosis.

Adaptor Protein Complex alpha Subunits↗
PubMed2004

Self-assembly of folic acid derivatives: induction of supramolecular chirality by hierarchical chiral structures.

Hierarchical chiral structures made up of dendritic oligo(L- or D-glutamic acid) moieties of folic acid derivatives induce supramolecular chirality in the self-assembled columnar structures of the folic acids. These folic acids self-assemble through the intermolecular hydrogen bonds of the pterin rings to form disklike tetramers. In the neat states, the stacked tetramers form thermotropic hexagonal columnar phases over wide temperature ranges, including room temperature. Addition of alkali metal salts induces chirality in the columnar phases. In dilute solution states in a relatively polar solvent (chloroform), the folic acid derivatives form non-chiral, self-assembled structures. In the presence of sodium triflate, the folic acid forms chiral columnar assemblies through the oligo(L-glutamic acid) moiety, similar to those formed in the liquid-crystalline (LC) states. The enantiomer of the folic acid induces columnar assemblies with reversed helicity. In the case of the diastereomer, no induced helicity is observed. Application of an apolar solvent (dodecane) drives the folic acid derivatives to form chiral assemblies in the absence of ions. In this case, lipophilic interactions promote nanophase segregation, which enhances the formation of chiral columns. Interestingly, the chiral supramolecular structure of the diastereomer induces the most intense circular dichroism. In both cases, the molecular chirality in the oligo(glutamate) moieties yields supramolecular chirality of the folic acids that self-assemble through cooperative molecular interactions.

Journal Article↗
PubMed2004

Two-component dendritic gel: effect of stereochemistry on the supramolecular chiral assembly.

The self-assembly of diaminododecane solubilised by four different stereoisomeric dendritic peptides to form gel-phase materials in toluene was investigated. The second generation dendritic peptides were based on D- and L-lysine building blocks, and each contained three chiral centres. By designing dendritic peptides in which the configurations of the chiral centres were modified, and applying them as gelator units, the assembly of stereoisomers could be investigated. In all cases, the self-assembly of gelator units resulted in macroscopic gelation. However, the degree of structuring was modulated by the stereoisomers employed, an effect which changed the morphology and macroscopic behavior of the self-assembled state. Enantiomeric (L,L,L or D,D,D) gelator units formed fibrous molecular assemblies, whilst the racemic gel (50 % L,L,L : 50 % D,D,D) formed a flat structure with a "woven" appearance. Gelator units based on L,D,D or D,L,L dendritic peptides also formed fibrous assemblies, but small-angle X-ray scattering indicated significant morphological differences were caused by the switch in chirality. Furthermore, the macroscopic stability of the gel was diminished when these peptides were compared with their L,L,L or D,D,D analogues. In this paper it is clearly shown that individual stereocentres, on the molecular level, are directly related to the helicity within the fibre. It is argued that the chirality controls the pattern of hydrogen bonding within the assembly, and hence determines the extent of fibre formation and the macroscopic gel strength.

Circular Dichroism↗
PubMed2004

The single neurofilament subunit of lamprey may need another element for filament assembly.

Regenerating axon tips in transected lamprey spinal cord contain dense accumulations of neurofilaments (NFs), suggesting that NFs may play a role in the mechanism of axonal regeneration. Compared with heteropolymeric assemblies of NF triplet proteins in mammals, NF in lampreys has been thought to contain only a single subunit (NF180). This would imply that NF180 self-assembles, which would be important for manipulating its expression in studies of axonal regeneration. In order to study the possible role of NF in process outgrowth and to determine whether NF180 can self-assemble, its gene was transfected into mammalian and fish cell lines that either contain or lack vimentin. In transfected NIH3T3 cells, NF180 was poorly phosphorylated and its expression did not alter the length or number of cell processes. Nor did it appear to form typical intermediate filaments, suggesting that it may not self-assemble. NF180 also did not form typical filaments in SW13cl cells that either possessed or lacked vimentin, nor in transfected fish cells that were cultured at 18 degrees C. In vitro, NF180 could not self-assemble but interacted with NF-L to interrupt its self-assembly. When cotransfected with rat NF-L into SW13c1.2vim(-) cells, NF180 did form thick, rod-like filamentous structures on immunofluorescence. More typical NFs were observed when NF180 was cotransfected with both NF-L and NF-M. Thus, NF180 cannot self-assemble but appears to require one or more additional elements for incorporation into NFs.

Animals↗
PubMed1991

Assembly, disassembly, and exchange of glial fibrillary acidic protein.

The kinetics and dynamics of glial fibrillary acidic protein (GFAP) assembly was explored by a fluorescence energy transfer assay method. Purified GFAP was stoichiometrically labeled at a single cysteine residue with fluorescein-maleimide. Soluble labeled GFAP in a low ionic strength buffer was assembled into 10 nm filaments by rapidly increasing the ionic strength, and the kinetics of GFAP assembly was monitored by the reduction in fluorescence due to self-quenching of fluorescein. The extent of fluorescence quench correlated with both the formation of 10 nm filament morphology and the amount of protein pelleted at 12,000g. The assembly of GFAP is critically dependent upon both protein and magnesium ion concentration, and at the critical concentration for GFAP assembly is approximately 40 micrograms/ml. Disassembly of GFAP filaments was also observed as a relief of fluorescence quenching after dilution of labeled GFAP filaments. When labeled GFAP filaments were mixed with an excess of unlabeled filaments, a rapid increase of fluorescence was observed, which is due to an exchange of subunits between labeled and unlabeled GFAP filaments. These results indicate that GFAP filaments are dynamic structures and that a small pool of kinetically active unassembled GFAP subunits are in a dynamic equilibrium with assembled GFAP filaments. The ability of GFAP to assemble, disassemble, and undergo subunit exchange has important implications for the organization and dynamics of astroglia cell cytoskeleton during development and in response to injury.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

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.

Adenosine Triphosphate↗