Disk aggregates of tobacco mosaic virus protein in solution: electron microscopy observations

Previous studies of the coat protein of tobacco mosaic virus (TMVP) have shown that TMVP presumably exists as linear stacks of two-ring cylindrical disks in the 0.7 M ionic strength buffer used for crystallizing the disks for X-ray diffraction studies [Raghavendra, K., Adams, M.L., & Schuster, T.M. (1985) Biochemistry 24, 3298-3304]. The spectroscopic and sedimentation studies of solutions of TMVP under these crystallizing conditions have demonstrated a long-term metastability of these disk aggregates when they are placed in 0.1 M ionic strength buffers, as are used for reconstituting tobacco mosaic virus from TMVP and viral RNA. The present work describes an electron microscopic study of TMVP disk aggregates under the same solution conditions employed in the previous spectroscopic and sedimentation studies. The results show that in the pH 8.0 0.7 M ionic strength crystallization buffer TMVP exists as stacks of disks which range in size from about 6 to 24 layers, corresponding to 3-12 2-layer disk aggregates having 17 subunits per layer. These TMVP aggregates persist in a metastable form in 0.1 M ionic strength virus reconstitution buffer with no apparent changes in structure of the stacked disks. The results are consistent with the conclusions of the solution physical-chemical studies which suggest that the disk structure may not be related to the 20S TMVP aggregate that is the nucleation species in virus     

Structure and function of disk aggregates of the coat protein of tobacco mosaic virus

Experiments have been carried out on the coat protein of tobacco mosaic virus (TMVP) to test for the occurrence of the previously postulated RNA-induced direct switching, during in vitro assembly of tobacco mosaic virus (TMV), of the subunit packing from the cylindrical bilayer disk to the virus helical arrangement. No evidence was found for such RNA-induced switching and no evidence for the direct participation of the bilayer disk in either the nucleation or elongation phases of the in vitro virus assembly. Instead, virus assembly proceeds by an initiation step involving the binding of the RNA to the previously characterized two-plus turn helical aggregate that is formed from small oligomers of subunits. However, a bilayer disk, which has been characterized in high ionic strength crystals, has been observed in low ionic strength virus assembly solutions only as a transient species upon depolymerization of dimers of bilayer disks formed in solution at high ionic strength, and not as an equilibrium species of TMVP.     

Temperature dependence of the structure of aggregates of tobacco mosaic virus protein at pH 7.2. Static synchrotron small-angle X-ray scattering

The small-angle X-ray scattering (SAXS) method using a synchrotron radiation source was applied to the study of the self-aggregation process of tobacco mosaic virus protein (TMVP) at a concentration of 5.0 or 12.0 mg ml-1 in 50 mM or 100 mM-phosphate buffer (ionic strengths approx. 0.1 and 0.2, respectively) at pH 7.2 in the temperature region of 4.8 to 25.0 degrees C. This paper presents the results of static measurements of SAXS. Sedimentation velocity experiments were performed simultaneously under the same conditions. These results are qualitatively parallel to those of the SAXS measurements, although the size of stacked disks derived from the SAXS measurements is larger than that derived from the sedimentation experiments, suggesting a change in the equilibrium conditions in the centrifugal field. Qualitative analysis of the SAXS data with model simulation calculations implies that the aggregation of TMVP consists of two steps: (1) the aggregation of A-protein comprising a few subunits to form double-layered disks; and (2) the random polymerization of double-layered disks by disk-stacking. Increase in temperature, ionic strength or protein concentration induced TMVP to polymerize to form a double-layered disk or a quadruple-layered short rod with consumption of A-proteins, accompanied by a small number of multi-layered short rods. The SAXS results indicate that the A-protein and the multilayered short rods are polydisperse with respect to size and shape, i.e. the mixture of A-protein, double-layered disks and multi-layered short rods coexists in the equilibrium state without pressure-induced partial dissociation of TMPV as observed during normal ultracentrifugation, and even under solution conditions in which the formation of double-layered disks or higher-order aggregates is favored.     

Dynamic mechanism of the self-assembly process of tobacco mosaic virus protein studied by rapid temperature-jump small-angle X-ray scattering using synchrotron radiation

The self-assembly process of tobacco mosaic virus protein (TMVP) was observed by rapid temperature-jump time-resolved solution X-ray small-angle scattering using synchrotron radiation. The temperature-jump device used for the X-ray measurements is rapid enough to cope with even the fastest-assembling process of TMVP, and accumulates data of reasonable signal-to-noise ratios with a minimum total counting time of 7.5 seconds. The measurements suggested that the 20 S disk of TMVP polymerized to stacked disks (short rods). The time to complete stacking varied from approximately 25 seconds to approximately 1200 seconds, depending on the solution condition and magnitude of the temperature gap. Higher protein concentration, ionic strength and temperature favoured faster association. The results were analysed in terms of a set of kinetic equations that describe the two-stage aggregation of TMVP with an equilibrium constant K1, and two rate constants k+2 and k-2 for association and dissociation of disks, respectively. The consistency of the analysis suggests that the TMVP assembly proceeds in two steps of: (1) the aggregation of A-proteins into double-layered disks; and (2) the stacking of double-layered disks. The kinetic analysis indicated that the stacking belongs to the lowest range of protein-protein interaction system.     

Effect of tubulin binding and self-association on the near-ultraviolet circular dichroic spectra of colchicine and analogues

Near-UV circular dichroic (CD) spectra of three colchicine analogues that differ at the C-10 position have been obtained in the presence and absence of tubulin. All three colchicine analogues show dramatic alterations in the low-energy near-UV CD band upon tubulin binding that cannot be mimicked by solvent, but in no event does the rotational strength of the CD band decrease to nearly zero as in the case of colchicine [Detrich, H. W., III, Williams, R. C., Jr., Macdonald, T. L., & Puett, D. (1981) Biochemistry 20, 5999-6005]. The effect of self-association of colchicine and one of the C-10 analogues, thiocolchicine, on the near-UV CD band was also investigated. A qualitative similarity was seen between the near-UV CD spectra of colchicine and thiocolchicine dimers and the spectra of these molecules bound to tubulin. These observations support the previous suggestion that ligands bound to the colchicine site on tubulin may be interacting with an aromatic amino acid in the colchicine binding site [Hastie, S. B., & Rava, R. P. (1989) J. Am. Chem. Soc. 110, 6993-7001].     

States of aggregation of the dahlemense strain of tobacco mosaic virus protein and their relation to crystal formation.

The aggregation of the protein of the dahlemense strain of tobacco mosaic virus has been studied by electron microscopy and ultracentrifugation. The aggregates formed are similar to those formed by the vulgare strain, although the particular conditions for their formation are often rather different. Helix formation by dialysis of A protein at pH 8 to acid pH is much more efficient if an intermediate step at pH 7 is introduced. The 20 S particle or two-layer disk is stable over a wide range of pH and ionic strength values. There is no tendency to form short stacks of disks at high ionic strength; instead, 30 S particles are formed that correspond to a pair of interlocked disks giving a “figure-of-eight” appearance in electron micrographs. These particles appear to be the “building blocks” of the protein crystal.     

Protease-sensitive scrapie prion protein in aggregates of heterogeneous sizes

The pathological prion protein PrP(Sc) is the only known component of the infectious prion. In cells infected with prions, PrP(Sc) is formed posttranslationally by the refolding of the benign cell surface glycoprotein PrP(C) into an aberrant conformation. The two PrP isoforms possess very different properties, as PrP(Sc) has a protease-resistant core, forms very large amyloidic aggregates in detergents, and is only weakly immunoreactive in its native form. We now show that prion-infected rodent brains and cultured cells contain previously unrecognized protease-sensitive PrP(Sc) varieties. In both ionic (Sarkosyl) and nonionic (n-octyl beta-D-glucopyranoside) detergents, the novel protease-sensitive PrP(Sc) species formed aggregates as small as 600 kDa, as measured by gel filtration. The denaturation dependence of PrP(Sc) immunoreactivity correlated with the size of the aggregate. The small PrP(Sc) aggregates described here are consistent with the previous demonstration of scrapie infectivity in brain fractions with a sedimentation coefficient as small as 40 S [Prusiner et al. (1980) J. Neurochem. 35, 574-582]. Our results demonstrate for the first time that prion-infected tissues contain protease-sensitive PrP(Sc) molecules that form low MW aggregates. Whether these new PrP(Sc) species play a role in the biogenesis or the pathogenesis of prions remains to be established.     

Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein

Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlations among morphology, beta-sheet stability, and molecular structure in prion peptide aggregates

The misfolding of proteins into beta-sheets and the subsequent aggregation of these sheets into fibrous networks underlies many diseases. In this paper, the role of peptide structure in determining the ordering of beta-sheet aggregates and the morphology of fibrils and protofibrils is dissected. Using a series of peptides based on residues 109-122 of the Syrian hamster prion protein (H1) with a range of substitutions at position 117, the link between side chain interactions and beta-sheet thermal stability has been investigated. The thermal stability of beta-sheets is associated with the peptides' ability to adopt the same alignment as wild-type H1, with residue 117 in register across all beta-strands [Silva, R. A. G. D., Barber-Armstrong, W., and Decatur, S. M. (2003) J. Am. Chem. Soc. 125, 13674-13675]. These aligned strands are capable of forming long, rigid, and twisted fibrils (as visualized by atomic force microscopy) which are thermostable. Peptides which do not adopt this strand alignment aggregate to form thin, flexible, and smooth protofibrils. The ability to form ordered aggregates, and thus to form twisted fibrils, is modulated by the structure of the side chain of residue 117.     

Cluster structure of anaerobic aggregates of an expanded granular sludge bed reactor

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH(4)/g of volatile suspended solids [VSS].day or 1.1 g of CH(4) chemical oxygen demand/g of VSS.day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens (Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (Methanobacterium-like and Methanospirillum-like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.     

Transformed mouse glucocorticoid receptor: generation and interconversion of the 3.8S, monomeric and 5.2S, oligomeric species

Recent studies have implicated subunit dissociation as a possible mechanism of glucocorticoid receptor transformation [Vedeckis, W.V. (1983) Biochemistry 22, 1983-1989; Raaka, B.M., & Samuels, H.H. (1983) J. Biol. Chem. 258, 417-425]. While it is becoming increasingly evident that the untransformed (non-nuclear-binding and non-DNA-binding) glucocorticoid receptor from mouse AtT-20 cells is a 9.1S oligomeric species (Mr 290 000-360 000), two transformed species have been described for this receptor. One of these has a sedimentation coefficient of 5.2 S (on molybdate-containing gradients), while the smallest nonproteolyzed, monomeric subunit is 3.8 S. The present study was undertaken to determine which is the most common form generated both in vitro and in vivo and the structural relationship between these two forms. A wide variety of in vitro transformation protocols all yielded the 5.2S form when analyzed on molybdate-containing sucrose gradients by using a vertical tube rotor. Kinetic studies showed that the appearance of the 5.2S form coincided precisely with the appearance of transformed receptor, as defined by DEAE-cellulose elution. Furthermore, when the 3.8S and 5.2S peaks were collected from sucrose gradients directly, they were transformed receptors as defined by both DEAE-cellulose and DNA-cellulose chromatography, while the 9.1S sucrose gradient peak was untransformed when the same criteria were used. The 3.8S monomer, when isolated from high-salt sucrose gradients and then desalted, reverted to the 5.2S form (molybdate-containing gradients) or a 6.6S form (low-salt, molybdate-free gradients).(ABSTRACT TRUNCATED AT 250 WORDS)     

Light stimulates phosphorylation of two large membrane proteins in frog photoreceptors

By photoactivating rhodopsin, light indirectly initiates a series of biochemical reactions within photoreceptors as part of the visual process. I herein report that one of the light-stimulated reactions in bullfrog photoreceptors is the phosphorylation of two previously unreported proteins (220 and 240 kDa). Their phosphorylation by endogenous kinase(s) is readily observed in freshly isolated, fragmented rods. On subcellular fractionation, the labeled proteins copurify with the membranes of the outer segments, from which they cannot be extracted with low ionic strength. They appear to be integral membrane proteins of the disk or plasma membranes. Their light-induced phosphorylation is also observed in intact receptors when excised frog retinas are incubated under in vivo conditions with 32PO4. Thus, appropriate kinase(s) is (are) present within outer segments and presumably is (are) the one(s) responsible for phosphorylation in fragmented cells. In the presence of adenosine 5'-(gamma-[35S]thiotriphosphate) [( 35S] ATP-gamma-S), light can also stimulate thiophosphorylation, leading to preferential labeling of the 220-kDa protein. On the basis of four criteria (electroporetic mobility, membrane location, binding of concanavalin A, and mobility shifts with SH oxidation), the 220-kDa protein appears to correspond to the membrane protein previously identified at the rims of rod disks [Papermaster, D.S., Schneider, B.G., Zorn, M.A. & Kraehenbuhl, J.P. (1978) J. Cell Biol. 78, 415-425]. Identity of the other substrate protein is unknown. When fragmented cells are illuminated with a flash of 1-ms duration, the half-time for phosphorylation is about 1 min with ATP at 0.1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H-NMR studies of high-potential iron-sulfur protein from the purple phototrophic bacterium, Rhodospirillum tenue

The high-potential iron-sulfur protein (HiPIP) from Rhodospirillum tenue (strain 3761) shows only a weak (20-25%) sequence similarity to HiPIPs from Chromatium vinosum, Ectothiorhodospira halophila and Ectothiorhodospira vacuolata, including the strict conservation of only two of the twelve residues assumed to be in the 4Fe-4S cluster packing region [Tedro, S. M., Meyer, T. E. and Kamen, M. D. (1979) J. Biol. Chem. 254, 1495-1500]. In spite of these differences, the general range and distribution of hyperfine-shifted 1H-NMR peaks of oxidized and reduced R. tenue HiPIP resemble those of E. halophila HiPIP I [Krishnamoorthi, R., Markley, J. L., Cusanovich, M. A., Pryzycieki, C. T. and Meyer, T. E. (1986) Biochemistry 25, 60-67]. Temperature- and pH-dependence and longitudinal relaxation behavior were determined for hyperfine-shifted peaks of the oxidized protein. Tentative assignments of peaks to ligands and aromatic residues suggest the presence of common apoprotein-active-site interactions in these proteins. Differences occur in the pattern of paramagnetically shifted peaks attributed to hydrogens bonded to the 4Fe-4S cluster. Hyperfine-shifted peaks of R. tenue HiPIP are not perturbed by pH changes in the range 5-9. In contrast, those of the C. vinosum protein exhibit a pH-dependence of chemical shifts that has been attributed to the titration of His42 [Nettesheim, D. G., Meyer, T. E., Feinberg, B. A. and Otvos, J. D. (1983) J. Biol. Chem. 258, 8235-8239]. Since R. tenue HiPIP contains no histidine, the present observation confirms the above hypothesis.     

Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein

The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.     

Role of histone tails in the conformation and interactions of nucleosome core particles

The goal of this work was to test the role of the histone tails in the emergence of attractive interactions between nucleosomes above a critical salt concentration that corresponds to the complete tail extension outside the nucleosome [Mangenot, S., et al (2002) Biophys. J. 82, 345-356; Mangenot, S., et al (2002) Eur. Phys. J. E 7, 221-231]. Small angle X-ray scattering experiments were performed in parallel with intact and trypsin tail-deleted nucleosomes with 146 +/- 3 bp DNA. We varied the monovalent salt concentration from 10 to 300 monovalent salt concentration and followed the evolution of (i) the second virial coefficient that characterizes the interactions between particles and (ii) the conformation of the particle. The attractive interactions do not emerge in the absence of the tails, which validates the proposed hypothesis.     

Cholesterol heterogeneity in bovine rod outer segment disk membranes

Rod outer segment disk membranes have been used to study visual transduction events. Numerous studies have also focused on protein-lipid interactions in these membranes. The possible heterogeneity of the disk membrane composition has not been addressed in such studies. Freeze fracture studies (Andrews, L. D., and Cohn, A. I. (1979) J. Cell Biol. 81, 215-220; Caldwell, R., and McLaughlin, B. (1985) J. Comp. Neurol. 236, 523-537) suggest a difference in cholesterol content between newly formed and old disks. This potential heterogeneity in disk membrane composition was investigated using digitonin. Osmotically intact bovine rod outer segment disk membranes prepared by Ficoll flotation were separated based on the cholesterol content of the disks. The addition of digitonin to disk membrane suspensions in a one-to-one molar ratio with respect to cholesterol produced an increase in the density of the membranes in proportion to the amount of cholesterol present. The digitonin-treated disks were separated into subpopulations using a sucrose density gradient. Disks were shown to vary in cholesterol to phospholipid ratio from 0.30 to 0.05. The ratio of phospholipid to protein remained constant in all disk subpopulations at approximately 65 phospholipids per protein. No significant change in the fatty acid composition of the disks was observed as a function of change in cholesterol content. This work demonstrates compositional heterogeneity in disk membranes which may ultimately affect function.     

Conversions of poly(2-aminodeoxyadenylate-5-halodeoxyuridylate) from B to A forms in high salt. An NMR and circular dichroism study

Proton one- and two-dimensional nuclear Overhauser enhancement (1D and 2D NOE) spectroscopy has been used to demonstrate that poly(d2NH2A-d5IU) and poly(d2NH2A-d5BrU) are converted from the B to the A conformation in high salt, as found previously for poly(d2NH2A-dT) [Borah, B., Cohen, J. S., Howard, F. B., & Miles, H. T. (1985) Biochemistry 24, 7456-7462]. The 2D NOE and 1D NOE spectra exhibit strong base proton (H8,H6)-H3' cross relaxation, suggesting short interproton distances. These results are indicative of a C3'-endo sugar pucker for both purine and pyrimidine residues in an A or closely related structure. The circular dichroism and UV spectra are consistent with the interpretation of an A conformation in high salt.     

Disulfide-bonded aggregates of heparan sulfate proteoglycans

Heparan sulfate proteoglycans have been isolated from Swiss mouse 3T3 cells by using two nondegradative techniques: extraction with 4 M guanidine or 2.5% 1-butanol. These proteoglycans were separated from copurifying chondroitin sulfate proteoglycans by using ion-exchange chromatography on DEAE-cellulose in the presence of 2 M urea. The purified heparan sulfate proteoglycans are substantially smaller, ca. Mr 20 000, than those isolated from these same cells with trypsin, ca. Mr 720 000 [Johnston, L.S., Keller, K. L., & Keller, J. M. (1979) Biochim. Biophys. Acta 583, 81-94]. However, all of the heparan sulfate proteoglycans extracted by these three methods contain similar glycosaminoglycan chains (Mr 7500) and are derived from the same pool of cell surface associated molecules. The trypsin-released heparan sulfate proteoglycan (ca. Mr 720 000) can be significantly reduced in size (ca. Mr 33 000) under strong denaturing conditions in the presence of the disulfide reducing agent dithiothreitol, which suggests that this form of the molecule is a disulfide-bonded aggregate. The heparan sulfate proteoglycan isolated from the medium also undergoes a significant size reduction in the presence of dithiothreitol, indicating that a similar aggregate is formed as part of the normal release of heparan sulfate proteoglycans into the medium. These results suggest that well-shielded disulfide bonds between individual heparan sulfate proteoglycan monomers may account for the large variation in sizes which has been reported for heparan sulfate proteoglycans isolated from a variety of cells and tissues with a variety of extraction procedures.     

Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes. 2. Changes in mechanism as a function of sodium chloride concentration and other solution variables

The dissociation kinetics of bacteriophage T4 coded gene 32 protein-single-stranded nucleic acid complexes have been examined as a function of monovalent salt concentration, temperature, and pH in order to investigate the details of the dissociation of cooperatively bound protein. Fluorescence stopped-flow techniques were used, and irreversible dissociation was induced by a combination of [NaCl] jumps and mixing with excess nucleic acid competitor. This made it possible to directly investigate the irreversible dissociation process over a wide range of NaCl concentrations [e.g., from 50 mM to 0.60 M for the gene 32 protein-poly(A) complex], in the absence of reassociation. Over the entire salt range, the only dissociable species observed is the singly contiguously bound gene 32 protein which dissociates from the ends of protein clusters. However, the [NaCl] dependence of the dissociation rate constant suggests that two competing pathways exist for dissociation of cooperatively bound gene 32 protein from the ends of protein clusters. At high monovalent salt concentrations, dissociation is dominated by a single-step process, with log ke/log [NaCl] = 6.5 +/- 0.5; i.e., the dissociation rate constant increases with increasing NaCl concentration due to the uptake of approximately six monovalent ions upon dissociation. This indicates that singly contiguous protein dissociates directly into solution. However, at much lower [NaCl] the data suggest that gene 32 protein, when bound at the end of a protein cluster, dissociates by first sliding off the end to form a noncooperatively bound intermediate which subsequently dissociates. A quantitative model which incorporates the sliding pathway [Berg, O. G., Winter, R. B., & von Hippel, P. H. (1981) Biochemistry 20, 6929-6948] in the dissociation mechanism fits the data reasonably well and suggests that noncooperatively bound monomers of gene 32 protein may be capable of one-dimensional translocation along single-stranded nucleic acids as suggested by independent kinetic data on the association reaction [Lohman, T. M., & Kowalczykowski, S. C. (1981) J. Mol. Biol. 152, 67-109]. It is also observed that both the absolute dissociation rate constant for T4 gene 32 protein and its salt dependence are sensitive to the average molecular weight and polydispersity of the nucleic acid sample used. This is a general phenomenon exhibited by proteins that bind to nucleic acids in a highly cooperative manner.     

Nucleosome rearrangement in vitro. 2. Formation of nucleosomes in newly repaired regions of DNA

We have reported previously that immediately following nucleotide excision repair in human cells the newly repaired DNA lacks a nucleosome conformation [Smerdon, M. J., & Lieberman, M. W. (1980) Biochemistry 19, 2992-3000]. In this study, we have examined the ability of these nascent DNA regions to acquire a nucleosome structure in vitro by incubating intact or H1-depleted nuclei in buffers containing different salt concentrations (0.025-0.625 M KCl) at 0 or 37 degrees C. Nucleosomes were detected in these regions by an increase in the level of repair-incorporated nucleotides associated with isolated nucleosome core particle DNA. Our results indicate that the nascent DNA is resistant to nucleosome formation during the low-salt transition where the limiting repeat length decreases from approximately 190 to 168 base pairs (bp) [Watkins, J. F., & Smerdon, M. J. (1985) Biochemistry (preceding paper in this issue)]. This result provides further evidence that the nascent DNA is indeed in a nonnucleosomal state. At higher salt concentrations (greater than 0.4 M), where the nucleosome repeat length decreases to a limiting value of approximately 146 bp, there was an increase in nucleosome formation in nascent DNA that correlated with the decrease in limiting repeat length. However, we did not observe a complete randomization of the repair-incorporated nucleotides. Indeed, even at the highest salt concentration used (0.625 M), we never observed more than 50% of the nascent DNA associated with the isolated core particles. This was the case even though a major portion of the nucleosomes had a limiting value repeat length following the high-salt incubation.(ABSTRACT TRUNCATED AT 250 WORDS)