The Renaturation of Denatured DNA

The kinetics of renaturation of heat-denatured DNA from E. coli and pneumococcus have been examined by ultraviolet absorption measurements. The molecularity of the reaction was assessed by three independent treatments of the data, and all lead to the conclusion that renaturation is essentially first order at 60°; at 70° and 80° there is an increasing second order component, resulting in simultaneous unimolecular and bimolecular kinetics. The unimolecular kinetics rule out reaction between two, kinetically separate strands, indicating rather the zippering-up of a single, denatured entity. The bimolecular kinetics can be attributed to the complexing of two such entities; thus, the genetic or density-labeled complexes that have been observed by other investigators can be accounted for without invoking strand separation. Since renaturation at best is never complete, the free ends of two renatured molecules permit sufficient bimolecular reaction to produce density hybrids. The observed kinetics can be accounted for if the hydrogen bonds of DNA are broken during heat denaturation but the strands do not separate. Light scattering supports this by showing that the molecular weight is unchanged by denaturation. Since there is no existing evidence that is inconsistent with this hypothesis, it is reasonable to conclude that heat denaturation does not completely separate the entangled strands of the DNA molecule.     

Renaturation and Hybridization Studies of Mitochondrial DNA

The products of the renaturation reaction of mitochondrial DNA from oocytes of Xenopus laevis have been studied by electron microscopy and CsCl equilibrium density gradient centrifugation. The reaction leads to the formation of intermediates containing single-stranded and double-stranded regions. Further reactions of these intermediates result in large complexes of interlinking double-stranded filaments. The formation of circular molecules of the same length as native circles of mitochondrial DNA was also observed. The formation of common high molecular weight complexes during joint reannealing of two DNA's with complementary sequences was used as a method to detect sequence homology in different DNA samples. Although this method does not produce quantitative data it offers several advantages in the present study. No homologies could be detected between the nuclear DNA and the mitochondrial DNA of X. laevis or of Rana pipiens. In interspecies comparisons homologies were found between the nuclear DNA's of X. laevis and the mouse and between the mitochondrial DNA's of X. laevis and the chick, but none between the mitochondrial DNA's of X. laevis and yeast. These results are interpreted as indicating the continuity of mitochondrial DNA during evolution.     

Renaturation of denatured DNA in NaI gradients

We show that denatured DNA from Tetrahymena mitochondria or phage T₇, partially renatures during centrifugation in NaI equilibrium density gradients. This makes these gradients unsuitable for the analysis of single-stranded complementary nucleic acids, especially if their complexity and mole percent G+C are low.     

Renaturation of DNA: a novel reaction of histones.

Histones isolated from several sources, either singly or in combination promote the renaturation of complementary single strands of DNA, as measured by the acquisition of resistance to S1 nuclease. The reaction is rapid (T1/2 less than 1 min), and is stoichiometric rather than catalytic. Renaturation is stimulated by Mg2+, Mn2+, and Ca2+, but is strongly inhibited by Zn2+. Crude extracts of early embryos of Drosophila melanogaster possess renaturation activity which is protease sensitive, heat-stable, and acid-soluble, suggesting that most or all of it can be attributed to histones. This observation thus provides a functional assay for histones that should prove useful in studies of chromatin and histone-DNA interactions, as well as for the identification and isolation of histones and histone-like proteins in crude extracts.     

Renaturation of copper solutions of DNA. I. Phenomenological analysis

A study of the kinetics of renaturation of cupric DNA solutions has been carried out by two physical properties methods. The use of a purely phenomenological analysis leads nevertheless to interesting conclusions concerning the transformation mechanism. We have found that: in the presence of Cu⁺⁺ ions, different. DNA samples exhibit an equivalent kinetic behavior; the system can be decomposed into two parts: DNA and copper ions, and the rate of formation of DNA should be faster than that of copper ions; the mechanism of transformation may be bimolecular, but further investigation is necessary to confirm this.     

DNA Motion Capture Reveals the Mechanical Properties of DNA at the Mesoscale

Single-molecule studies probing the end-to-end extension of long DNAs have established that the mechanical properties of DNA are well described by a wormlike chain force law, a polymer model where persistence length is the only adjustable parameter. We present a DNA motion-capture technique in which DNA molecules are labeled with fluorescent quantum dots at specific sites along the DNA contour and their positions are imaged. Tracking these positions in time allows us to characterize how segments within a long DNA are extended by flow and how fluctuations within the molecule are correlated. Utilizing a linear response theory of small fluctuations, we extract elastic forces for the different, ∼2-μm-long segments along the DNA backbone. We find that the average force-extension behavior of the segments can be well described by a wormlike chain force law with an anomalously small persistence length.     

Renaturation kinetics and thermal stability of DNA in aqueous solutions of formamide and urea.

This paper reports the results of a systematic study of the effects of formamide and urea on the thermal stability and renaturation kinetics of DNA. Increasing concentrations of urea in the range 0 to 8 molar lower the Tm by 2.25 degrees C per molar, and decreases the renaturation rate by approximately 8 percent per molar. Increasing concentrations of formamide in the range from 0 to 50 percent lowers the Tm by 0.60 degrees C per percent formamide for sodium chloride concentrations ranging from 0.035M to 0.88M. At higher salt concentrations the dependence of Tm on percent formamide was found to be slightly greater. Increasing formamide concentration decreases the renaturation rate linearly by 1.1% per percent formamide such that the optimal rate in 50% formamide is 0.45 the optimal rate in an identical solution with no formamide. The effects of urea and formamide on the renaturation rates of DNA are explained by consideration of the viscosities of the solutions at the renaturation temperatures.     

Mesoscale Liquid Model of Chromatin Recapitulates Nuclear Order of Eukaryotes

The nuclear envelope segregates the genome of Eukaryota from the cytoplasm. Within the nucleus, chromatin is further compartmentalized into architectures that change throughout the lifetime of the cell. Epigenetic patterns along the chromatin polymer strongly correlate with chromatin compartmentalization and, accordingly, also change during the cell life cycle and at differentiation. Recently, it has been suggested that subnuclear chromatin compartmentalization might result from a process of liquid-liquid phase separation orchestrated by the epigenetic marking and operated by proteins that bind to chromatin. Here, we translate these observations into a diffuse interface model of chromatin, which we named the mesoscale liquid model of nucleus. Using this streamlined continuum model of the genome, we study the large-scale rearrangements of chromatin that happen at different stages of the growth and senescence of the cell and during nuclear inversion events. In particular, we investigate the role of droplet diffusion, fluctuations, and heterochromatin-lamina interactions during nuclear remodeling. Our results indicate that the physical process of liquid-liquid phase separation, together with surface effects, is sufficient to recapitulate much of the large-scale morphology and dynamics of chromatin along the life cycle of cells.     

Minimal Mesoscale Model for Protein-Mediated Vesiculation in Clathrin-Dependent Endocytosis

In eukaryotic cells, the internalization of extracellular cargo via the endocytic machinery is an important regulatory process required for many essential cellular functions. The role of cooperative protein-protein and protein-membrane interactions in the ubiquitous endocytic pathway in mammalian cells, namely the clathrin-dependent endocytosis, remains unresolved. We employ the Helfrich membrane Hamiltonian together with surface evolution methodology to address how the shapes and energetics of vesicular-bud formation in a planar membrane are stabilized by presence of the clathrin-coat assembly. Our results identify a unique dual role for the tubulating protein epsin: multiple epsins localized spatially and orientationally collectively play the role of a curvature inducing capsid; in addition, epsin serves the role of an adapter in binding the clathrin coat to the membrane. Our results also suggest an important role for the clathrin lattice, namely in the spatial- and orientational-templating of epsins. We suggest that there exists a critical size of the coat above which a vesicular bud with a constricted neck resembling a mature vesicle is stabilized. Based on the observed strong dependence of the vesicle diameter on the bending rigidity, we suggest that the variability in bending stiffness due to variations in membrane composition with cell type can explain the experimentally observed variability on the size of clathrin-coated vesicles, which typically range 50–100 nm. Our model also provides estimates for the number of epsins involved in stabilizing a coated vesicle, and without any direct fitting reproduces the experimentally observed shapes of vesicular intermediates as well as their probability distributions quantitatively, in wildtype as well as CLAP IgG injected neuronal cell experiments. We have presented a minimal mesoscale model which quantitatively explains several experimental observations on the process of vesicle nucleation induced by the clathrin-coated assembly prior to vesicle scission in clathrin dependent endocytosis.     

Renaturation of Bacillus thermoglucosidasius HrcA repressor by DNA and thermostability of the HrcA-DNA complex in vitro

HrcA, a negative control repressor for chaperone expression from the obligate thermophile Bacillus thermoglucosidasius KP1006, was purified in a His-tagged form in the presence of 6 M urea but hardly renatured to an intact state due to extreme insolubility. Renaturation trials revealed that the addition of DNA to purified B. thermoglucosidasius HrcA can result in solubilization of HrcA free from the denaturing agent urea. Results from band shift and light scattering assays provided three new findings: (i) any species of DNA can serve to solubilize B. thermoglucosidasius HrcA, but DNA containing the CIRCE (controlling inverted repeat of chaperone expression) element is far more effective than other nonspecific DNA; (ii) B. thermoglucosidasius HrcA renatured with nonspecific DNA bound the CIRCE element in the molecular ratio of 2.6:1; and (iii) B. thermoglucosidasius HrcA binding to the CIRCE element was stable at below 50 degrees C whereas the complex was rapidly denatured at 70 degrees C, suggesting that the breakdown of HrcA is induced by heat stress and HrcA may act as a thermosensor to affect the expression of heat shock regulatory genes. These results will help to determine the nature of HrcA protein molecules.     

Renaturation of complementary DNA strands by herpes simplex virus type 1 ICP8.

ICP8, the major single-stranded DNA-binding protein of herpes simplex virus type 1, promotes renaturation of complementary single strands of DNA. This reaction is ATP independent but requires Mg2+. The activity is maximal at pH 7.6 and 80 mM NaCl. The major product of the reaction is double-stranded DNA, and no evidence of large DNA networks is seen. The reaction occurs at subsaturating concentrations of ICP8 but reaches maximal levels with saturating concentrations of ICP8. Finally, the renaturation reaction is second order with respect to DNA concentration. The ability of ICP8 to promote the renaturation of complementary single strands suggests a role for ICP8 in the high level of recombination seen in cells infected with herpes simplex virus type 1.     

Renaturation and interaction of ribonuclease A with AOT surfactant in reverse micelles

This study extended works on effects of solute on the percolation of reverse micelles to the effects of interactions between protein and surfactants on protein refolding by reverse micelles. The changes in percolation behavior were identified and attributed to the position of solutes in the core aqueous phase and the interaction between the solute and the surfactants. The percolation behavior of reverse micelles with solutes was related to protein renaturation and the reverse micelle. This study aims to highlight the involvement of the interface and the interaction of the protein with the surfactant during protein refolding. Ribonuclease A and AOT reverse micelles together constitute a model system considered here. The systemic parameters of the reverse micelle, water content (W(o)) and pH value, were applied to modify the interaction between the denatured protein molecules and the surfactant interface. The interactions and the locations of the protein molecules were determined from changes in percolation temperature measured by conductivity. The percolation and protein activity show that a stronger interaction of the protein molecules with surfactant corresponds to superior recovery of protein activity. The investigation concludes that the refolding of protein by reverse micelles is not only facilitated by the isolation of reverse micelles but also by the interaction due to the interface of the reverse micelle.     

A Kronig-Penney Model of Salts of DNA

A one dimensional Kronig-Penney model for a salt like Na DNA is given. The helical periodicity is treated in a manner suggested by Tinoco and Woody. Using data on the semiconductor band gap, we estimate the strength of the potential barrier. The energy limits of the ten bands filled by 20π electrons per unit cell are calculated and exhibited in Table I.     

Renaturation of reduced ribonuclease A with a microsphere-induced refolding system

We intended to refold reduced ribonuclease A (RNase A) using polymeric microspheres. Polymeric microspheres were allowed to react with dithiothreitol (DTT) to immobilize the disulfide and thiol moieties on their surface. The fully reduced RNase A was added to the dispersion of the modified microspheres. Protein refolding and renaturation were estimated by the change in the number of disulfide bonds of RNase A and the recovery of the enzymatic activity, respectively. Without microspheres, the activity gradually recovered with the increase in the number of disulfide bonds. However, the formation of disulfide bonds of reduced RNase A was accelerated by adding the modified microspheres, and the rate of renaturation was increased depending on the amount of charged DTT and the reaction time of the immobilization. These results indicate that modified microspheres significantly catalyze the recovery of active RNase A from the reduced form. The protein adsorption data demonstrated that the disulfide moieties of the modified microspheres react with the thiol moieties of the reduced RNase A to form a mixed disulfide. The thiol/disulfide exchange reaction can possibly proceed at the microsphere/protein interface, resulting in the formation of a correct three-dimensional structure.