Chromatin condensation dynamics and implications of induced premature chromosome condensation

Somatic cell cycle is a dynamic process with sequential events that culminate in cell division. Several physiological activities occur in the cytoplasm and nucleus during each of the cell cycle phases which help in doubling of genetic content, organized arrangement of the duplicated genetic material and perfect mechanism for its equal distribution to the two daughter cells formed. Also, the cell cycle checkpoints ensure that the genetic material is devoid of damages thus ensuring unaltered transmission of genetic information. Two important phenomena occurring during the cell cycle are the DNA condensation and decondensation cycles in the nucleus along with the cyclic expression and functioning of certain specific proteins that help in the same. Several protein families including Cyclins, cyclin dependent kinases, condensins, cohesins and surivins ensure error free, stage specific DNA condensation and decondensation by their highly specific, controlled orchestrated presence and action. Understanding the molecular mechanisms of chromatin compaction towards formation of the structural units, the chromosomes, give us valuable insights into the cellular physiology and also direct us to techniques such as premature chromosome condensation. The techniques of inducing ‘prophasing’ of interphase cells are undergoing rapid advances which have multidimensional applications for basic research and direct applications.     

Dynamics of DNA condensation

The condensation of DNA induced by spermine and spermidine is investigated by equilibrium titrations and stopped-flow and field-jump experiments using scattered light detection. The spermine concentration required for the cooperative condensation process is measured at different DNA concentrations; these data are used to evaluate both the condensation threshold degree of spermine binding and the binding constant of spermine according to an excluded-site model. Stopped-flow measurements of the spermine-induced condensation demonstrate the existence of two processes: (1) A "fast" reaction is observed in the millisecond time range, when the reactant concentrations are around 1 microM; it is associated with a characteristic induction period and is assigned to the intramolecular condensation reaction. (2) A slow reaction with time constants of, e.g., 100 s strongly dependent upon both spermine and DNA concentrations is assigned to an intermolecular DNA association. The unusual time course of the intramolecular condensation reaction with the induction period provides evidence for a "threshold kinetics". During the induction period, spermine molecules are bound to DNA, but the degree of binding remains below the threshold value. As soon as the degree of ligand binding arrives at the threshold, the DNA is condensed in a relatively fast reaction. Model calculations of the spermine binding kinetics according to an excluded-site model demonstrate that the spermine molecules bound to DNA are mobile along the double helix. A comparison of the experimental data with the results of Monte Carlo simulations suggests a rate constant of approximately 200 s-1 for spermine movement by one nucleotide residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative assessment of DNA condensation

A fluorescent method is proposed for assessing DNA condensation in aqueous solutions with variety of condensing agents. The technique is based on the effect of concentration-dependent self-quenching of covalently bound fluorophores upon DNA collapse. The method allows a more precise determination of charge equivalency in titration experiments with various polycations. The technique's ability to determine the number of DNA molecules that are condensed together in close proximity is under further investigation.     

Simple simulations of DNA condensation

Molecular dynamics simulations of a simple, bead-spring model of semiflexible polyelectrolytes such as DNA are performed. All charges are explicitly treated. Starting from extended, noncondensed conformations, condensed structures form in the simulations with tetravalent or trivalent counterions. No condensates form or are stable for divalent counterions. The mechanism by which condensates form is described. Briefly, condensation occurs because electrostatic interactions dominate entropy, and the favored coulombic structure is a charge-ordered state. Condensation is a generic phenomenon and occurs for a variety of polyelectrolyte parameters. Toroids and rods are the condensate structures. Toroids form preferentially when the molecular stiffness is sufficiently strong.     

Mitotic chromosome structure and condensation

Mitotic chromosome structure has been the cell biology equivalent of a 'riddle, wrapped in a mystery, inside an enigma'. Observations that genetic knockout or knockdown of condensin subunits or topoisomerase II cause only minimal perturbation in overall chromosome condensation, together with analysis of early stages of chromosome condensation and effects produced by histone H1 depletion, suggest a need to reconsider textbook models of mitotic chromosome condensation and organization.     

Counterion condensation in polyelectrolyte theory

Condensation of counterions to a single, highly charged polyelectrolyte is formulated in terms of the two-dimensional coulomb gas theory of Kosterlitz and Thouless. The critical condensation criterion is calculated and is shown to vary as a function of the added salt concentration.     

DNA condensation by multivalent cations

In the presence of multivalent cations, high molecular weight DNA undergoes a dramatic condensation to a compact, usually highly ordered toroidal structure. This review begins with an overview of DNA condensation : condensing agents, morphology, kinetics, and reversibility, and the minimum size required to form orderly condensates. It then summarizes the statistical mechanics of the collapse of stiff polymers, which shows why DNA condensation is abrupt and why toroids are favored structures. Various ways to estimate or measure intermolecular forces in DNA condensation are discussed, all of them agreeing that the free energy change per base pair is very small, on the order of 1% of thermal energy. Experimental evidence is surveyed showing that DNA condensation occurs when about 90% of its charge is neutralized by counterions. The various intermolecular forces whose interplay gives rise to DNA condensation are then reviewed. The entropy loss upon collapse of the expanded wormlike coil costs free energy, and stiffness sets limits on tight curvature. However, the dominant contributions seem to come from ions and water. Electrostatic repulsions must be overcome by high salt concentrations or by the correlated fluctuations of territorially bound multivalent cations. Hydration must be adjusted to allow a cooperative accommodation of the water structure surrounding surface groups on the DNA helices as they approach. Undulations of the DNA in its confined surroundings extend the range of the electrostatic forces. The condensing ions may also subtly modify the local structure of the double helix. © 1998 John Wiley & Sons, Inc. Biopoly 44: 269–282, 1997     

Epimerization in peptide thioester condensation

Peptide segment couplings are now widely utilized in protein chemical synthesis. One of the key structures for the strategy is the peptide thioester. Peptide thioester condensation, in which a C‐terminal peptide thioester is selectively activated by silver ions then condensed with an amino component, is a powerful tool. But the amino acid adjacent to the thioester is at risk of epimerization. During the preparation of peptide thioesters by the Boc solid‐phase method, no substantial epimerization of the C‐terminal amino acid was detected. Epimerization was, however, observed during a thioester–thiol exchange reaction and segment condensation in DMSO in the presence of a base. In contrast, thioester–thiol exchange reactions in aqueous solutions gave no epimerization. The epimerization during segment condensation was significantly suppressed with a less polar solvent that is applicable to segments in thioester peptide condensation. These results were applied to a longer peptide thioester condensation. The epimer content of the coupling product of 89 residues was reduced from 27% to 6% in a condensation between segments of 45 and 44 residues for the thioester and the amino component, respectively. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Mitotic chromosome condensation in vertebrates

Work from several laboratories over the past 10-15 years has revealed that, within the interphase nucleus, chromosomes are organized into spatially distinct territories [T. Cremer, C. Cremer, Chromosome territories, nuclear architecture and gene regulation in mammalian cells, Nat. Rev. Genet. 2 (2001) 292-301 and T. Cremer, M. Cremer, S. Dietzel, S. Muller, I. Solovei, S. Fakan, Chromosome territories-a functional nuclear landscape, Curr. Opin. Cell Biol. 18 (2006) 307-316]. The overall compaction level and intranuclear location varies as a function of gene density for both entire chromosomes [J.A. Croft, J.M. Bridger, S. Boyle, P. Perry, P. Teague,W.A. Bickmore, Differences in the localization and morphology of chromosomes in the human nucleus, J. Cell Biol. 145 (1999) 1119-1131] and specific chromosomal regions [N.L. Mahy, P.E. Perry, S. Gilchrist, R.A. Baldock, W.A. Bickmore, Spatial organization of active and inactive genes and noncoding DNA within chromosome territories, J. Cell Biol. 157 (2002) 579-589] (Fig. 1A, A'). In prophase, when cyclin B activity reaches a high threshold, chromosome condensation occurs followed by Nuclear Envelope Breakdown (NEB) [1]. At this point vertebrate chromosomes appear as compact structures harboring an attachment point for the spindle microtubules physically recognizable as a primary constriction where the two sister chromatids are held together. The transition from an unshaped interphase chromosome to the highly structured mitotic chromosome (compare Figs. 1A and B) has fascinated researchers for several decades now; however a definite picture of how this process is achieved and regulated is not yet in our hands and it will require more investigation to comprehend the complete process. From a biochemical point of view a vertebrate mitotic chromosomes is composed of DNA, histone proteins (60%) and non-histone proteins (40%) [6]. I will discuss below what is known to date on the contribution of these two different classes of proteins and their co-operation in establishing the final mitotic chromosome structure.     

Ion condensation and signal transduction

Many abiotic and other signals are transduced in eukaryotic cells by changes in the level of free calcium via pumps, channels and stores. We suggest here that ion condensation should also be taken into account. Calcium, like other counterions, is condensed onto linear polymers at a critical value of the charge density. Such condensation resembles a phase transition and has a topological basis in that it is promoted by linear as opposed to spherical assemblies of charges. Condensed counterions are delocalised and can diffuse in the so-called near region along the polymers. It is generally admitted that cytoskeletal filaments, proteins colocalised with these filaments, protein filaments distinct from cytoskeletal filaments, and filamentous assemblies of other macromolecules, constitute an intracellular macromolecular network. Here we draw attention to the fact that this network has physicochemical characteristics that enable counterion condensation. We then propose a model in which the feedback relationships between the condensation/decondensation of calcium and the activation of calcium-dependent kinases and phosphatases control the charge density of the filaments of the intracellular macromolecular network. We show how condensation might help mediate free levels of calcium both locally and globally. In this model, calcium condensation/decondensation on the macromolecular network creates coherent patterns of protein phosphorylation that integrate signals. This leads us to hypothesize that the process of ion condensation operates in signal transduction, that it can have an integrative role and that the macromolecular network serves as an integrative receptor.     

Bose-Einstein condensation in biological systems

A theoretical model is presented for describing a previously untreated effect of viscosity on the apparent decomposition rate of enzyme-ligand complexes.Since the translational diffusion is hindered by the viscosity, its increased value results in an enlarged portion of ligands which can be rebound by the enzyme immediately after the dissociation of the complex.The model accounts for the experimentally observed decrease in maximal velocity of enzymic reactions at high viscosity. At the same time, it serves as a tool to obtain new information about the energetic processes of enzyme action.     

B-chromosome condensation inPhleum pollen grains

Pollen grains were investigated in 11 specimens ofPhleum Boehmeri Wib. [P. phleoides (L.) Karst.] differing in the number of B-chromosomes (2n=14+0–3B). In some uninucleate pollen grains there were seen large chromocentres, not observed in other tissues. They resembled meiotically and mitotically condensed Bs both in size and shape. They occurred only in plants with 2 or 3 Bs. In the plants with 2 Bs, 2.5% pollen grains had a nucleus with 1 chromocentre; in the plants with 3 Bs, 41.4% of uninucleate pollen grains had 1 chromocentre and 0.2% of grains had 2 chromocentres. Comparison of the frequency of Bs in the metaphase of the first pollen mitosis and that of chromocentres in the nuclei of the uninucleate pollen grains was carried out. The author suggests that in pollen-grain nuclei one B chromosome per genome remains decondensed, and every next one (if it occurs) forms a chromocentre.     

Why double-stranded RNA resists condensation

The addition of small amounts of multivalent cations to solutions containing double-stranded DNA leads to inter-DNA attraction and eventual condensation. Surprisingly, the condensation is suppressed in double-stranded RNA, which carries the same negative charge as DNA, but assumes a different double helical form. Here, we combine experiment and atomistic simulations to propose a mechanism that explains the variations in condensation of short (25 base-pairs) nucleic acid (NA) duplexes, from B-like form of homopolymeric DNA, to mixed sequence DNA, to DNA:RNA hybrid, to A-like RNA. Circular dichroism measurements suggest that duplex helical geometry is not the fundamental property that ultimately determines the observed differences in condensation. Instead, these differences are governed by the spatial variation of cobalt hexammine (CoHex) binding to NA. There are two major NA-CoHex binding modes—internal and external—distinguished by the proximity of bound CoHex to the helical axis. We find a significant difference, up to 5-fold, in the fraction of ions bound to the external surfaces of the different NA constructs studied. NA condensation propensity is determined by the fraction of CoHex ions in the external binding mode.