Circular differential scattering and circular differential absorption of DNA-protein condensates and of dyes bound to DNA-protein condensates

DNA-protein condensates that give positive and negative psi-type circular dichroism (CD) spectra (psi condensates) bind intercalative and nonintercalative dyes. CD depends both on circular differential scattering and on circular differential absorption; scattering-corrected CD measurements are approximations to circular differential absorption. The circular differential scattering and scattering-corrected CD patterns observed in the DNA absorption band of psi condensates are mimicked in the induced CD band of intercalators bound to psi condensates. The induced scattering-corrected CD and circular differential scattering patterns of the groove-binding dye Hoechst 33342 bound to psi condensates are the inverse of the patterns seen with intercalative dyes, whereas the groove-binding dye manganese(III) meso-tetrakis(4-N-methylpyridyl)porphine [MnIIITMpyP-4] shows no significant induced CD patterns. The large circular differential scattering and scattering-corrected CD bands are interpreted as resulting from long-range chiral packing, rather than near-neighbor short-range interactions. Dyes intercalated into the DNA of the psi condensates have the same type of long-range chiral packing as the DNA bases. Therefore, the psi-type CD spectra seen in the UV spectra originating from the long-range packing of the DNA bases are also observed in the visible spectra when dyes are intercalated in the DNA of the psi condensates. Our interpretation comes from the observation that the induced circular differential scattering and circular differential absorption of the dye bound to the psi condensates depend only upon the sign of the circular differential absorption and the pattern of the circular differential scattering of the psi condensates without bound dye.(ABSTRACT TRUNCATED AT 250 WORDS)     

Watersoluble synthetic nucleic acid analogs--polyethyleneimine derivatives containing nucleic acid bases--conformation and interactionwith nucleic acids

Water soluble polyethyleneimine derivatives containing nucleic acid bases were found to interact with polynucleotides, DNA, RNA. The conformational change by formation of complex was observed by CD spectra and was discussed with the hypochromicity in UV spectra. The rates of interactions between nucleic acid bases in polymers were slow as shown by UV spectra, but the conformational changes of the polynucleotides were fast as shown by CD spectra. In the case of the uracil derivative (PEI-Hse-Ura), high value of CD spectra [theta] 2.80 = -8.0 x 10(-4) for the complex with DNA might be caused by psi type conformation of DNA.     

Study of DNA-spermine interactions by use of small-angle and wide-angle X-ray scattering and circular dichroism.

Circular dichroism measurements with DNA-spermine complexes at 0.075 M NaCl and at 0.15 M NaCl reveal +psi (type I) and -psi (type II) CD spectra respectively. From small-angle X-ray scattering studies it could be shown that type I has a long-range order, short-range order supramolecular structure, while type II is of long-range disorder, short-range disorder structure. The secondary structure of the DNA in both types of condensates is B-like as concluded from wide-angle X-ray scattering diagrams of the condensates and from a comparison with the wide-angle X-ray curves of DNA and RNA in solution.     

A comparison of small circular DNA molecules in psi+ and psi- strains of Saccharomyces cerevisiae.

The psi+ and psi- phenotypes, which affect the efficiency of ochre suppression in yeast, are inherited in a non-Mendelian fashion. There is no apparent difference in length or in length distribution of 2 micronm circular DNA molecules between psi+ and psi- strains. It seems that the psi genetic determinant is probably not connected with the presence or absence of these small circular DNA molecules.     

Membrane potential of E. coli recipient cells determines the rate of linear transport of DNA during conjugation

The rate of conjugal DNA transport from donor to recipient cells has been shown to depend on the membrane potential (delta psi) value in the DNA recipient cell. On the other hand, delta psi in the DNA donor cells is required for the formation of stable aggregates of conjugating cells, but not for the RNA transport. Both components of the electrochemical proton gradient on the cytoplasmic membrane of the recipient cells, the delta psi and the pH gradient are equivalent in the conjugal process.     

Condensation of DNA by the C-terminal domain of histone H1. A circular dichroism study

The condensation of DNA by the C-terminal domain of histone H1 has been studied by circular dichroism in physiological salt concentration (0.14 M NaF). As the intact H1 molecule, its C-terminal domain induces the so-called psi state of DNA that is characterized by a nonconservative circular dichroism spectrum which is currently attributed to ordered aggregation of the DNA molecules. On a molar basis, intact H1 and its C-terminal domain give spectra of similar intensity. Neither the globular domain of H1 nor an N-terminal fragment, that includes both the globular and N-terminal domains, has any effect on the conservative circular dichroism of DNA. From these results it is concluded that the condensation of DNA mediated by histone H1 is mainly due to its C-terminal domain. The effect of the salt concentration and the size of DNA molecules on the circular dichroism of the complexes are also examined.     

Devoted to the lagging strand-the subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly.

Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the beta sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called gamma complex that uses ATP to assemble the beta clamp onto DNA. We examine here the function of the psi subunit of the gamma complex clamp loader. Omission of psi from the holoenzyme prevents contact with single-stranded DNA-binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt. We also show that the product of a classic point mutant of SSB, SSB-113, lacks strong affinity for psi and is defective in promoting clamp loading and processive replication at elevated ionic strength. SSB-113 carries a single amino acid replacement at the penultimate residue of the C-terminus, indicating the C-terminus as a site of interaction with psi. Indeed, a peptide of the 15 C-terminal residues of SSB is sufficient to bind to psi. These results establish a role for the psi subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB-coated ssDNA.     

Topology of Xer recombination on catenanes produced by lambda integrase.

Xer site-specific recombination at the psi site from plasmid pSC101 displays topological selectivity, such that recombination normally occurs only between directly repeated sites on the same circular DNA molecule. This intramolecular selectivity is important for the biological role of psi, and is imposed by accessory proteins PepA and ArcA acting at accessory DNA sequences adjacent to the core recombination site. Here we show that the selectivity for intramolecular recombination at psi can be bypassed in multiply interlinked catenanes. Xer site-specific recombination occurred relatively efficiently between antiparallel psi sites located on separate rings of right-handed torus catenanes containing six or more nodes. This recombination introduced one additional node into the catenanes. Antiparallel sites on four-noded right-handed catenanes, the normal product of Xer recombination at psi, were not recombined efficiently. Furthermore, parallel psi sites on right-handed torus catenanes were not substrates for Xer recombination. These findings support a model in which psi sites are plectonemically interwrapped, trapping a precise number of supercoils that are converted to four catenation nodes by Xer strand exchange.     

水柱法场扩大提高CE-UV检测核酸灵敏度

目的：采用水柱法场扩大堆积技术,提高毛细管电泳紫外分析核酸灵敏度。方法：已知浓度的DNA Marker为标准样品,TE缓冲液递度稀释,压力进样前加一段去离子水柱（0.5psi,20s）,观察不同浓度下压力进样紫外检测图谱。结果：在对分离度无明显影响下,将压力进样时间延长至0.5psi,990s,与常规的压力进样（0.5psi,10s）相比,灵敏度提高了94.4倍。与时间延长后的压力进样（0.5psi,90s）相比,灵敏度提高8.2倍,最低检测总浓度为1ng/μl,DNA的检测限降至80ng/ml（S/N=3）,比前人报道的7ng/μl检测限提高了87.5倍。结论：验证了水柱法场扩大堆积注射可以有效提高毛细管电泳紫外检测核酸灵敏度。     

A function for the psi subunit in loading the Escherichia coli DNA polymerase sliding clamp

Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, chi and psi, which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, gamma(3)deltadelta', gamma(3)deltadelta'psi, and gamma(3)deltadelta'psichi. The psi subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the chi subunit does not contribute directly to clamp loading in our assays lacking single-stranded DNA-binding protein. The psi subunit also increases the affinity of the clamp loader for the clamp in assays in which ATPgammaS is substituted for ATP. Interestingly, the affinity of the gamma(3)deltadelta' complex for beta is no greater in the presence than in the absence of ATPgammaS. A role for psi in stabilizing or promoting ATP- and ATPgammaS-induced conformational changes may explain why large conformational differences were not seen in gamma(3)deltadelta' structures with and without bound ATPgammaS. The beta clamp partially compensates for the activity of psi when this subunit is not present and possibly serves as a scaffold on which the clamp loader adopts the appropriate conformation for DNA binding and clamp loading. Results from our work and others suggest that the psi subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding.     

Chromatin models. The ionic strength dependence of model histone-DNA interactions: circular dichroism studies of lysine-leucine polypeptide-DNA complexes.

The ionic strength dependence of the complexes between DNA and both random, (Lysx, Leuy)n, and block copolymers, (Lysx)n(Leuy)m, of lysine and leucine, with different amino acid compositions, was studied using circular dichroism (CD) as the probe to detect conformational differences in these complexes relative to native DNA. It was found that the CD spectra of complexes of both the random (Lys84, Leu16)n and block (Lys85)n(Leu15)m copolymers with DNA show a very sharp ionic strength dependence. The maximum altered CD spectrum for the complexes with the block copolymer was found to occur at the same ionic strength as that for poly(L-lysine)-DNA complexes, while the maximum CD change for the random copolymer complex occurred at a slightly lower ionic strength. This sharp dependence of the CD change on the ionic strength was found to be independent of the polymer/DNA ratio, r, for each individual copolymer. The CD spectra for these complexes at optimum NaCl concentration resemble those of the psi spectra of DNA [Jordan, C. F., Lerman, L.S., and Venable, J.H. (1972), Nature (London), New Biol. 236, 67]. The complexes of the random copolymer, (Lys68, Leu32)n, with DNA (r=0.25) at 0.15 M NaCl and below have CD spectra that resemble the A-form DNA spectra. The ionic strength dependence of the CD spectra of this complex is not as sharp as observed with the above polymers and has a broad positive plateau. It is suggested that both the CD spectra of these complexes reflect the phenomena of DNA condensation into a higher order asymmetric structure (folded and compact). The block copolymer, (Lys77)n(Leu23)m, complexes with DNA show very slight alterations in the CD spectra, with respect to native DNA. It appears that the long Leu sequence at one end of such copolymers may be unpropitious for causing the polypeptide-DNA complex to condense into a higher order asymmetric structure. Thus the importance of the distribution of hydrophobic residues, in the copolypeptides of Lys, is shown for causing condensation of complexes with DNA. The relevance of these findings to histone-DNA complexes in chromatin is discussed.     

Structural transitions of calf thymus DNA in concentrated LiCl solutions

The solubility, sedimentation, circular dichroism, and absorption spectral characteristics of calf thymus DNA have been examined in concentrated solutions of LiCl (6-13 m) at 25 to 27 degree C. At all concentrations of LiCl, the DNA is base stacked and exhibits normal hypochromicty, At the upper end of this range of LiCl concentrations, DNA aggregates and ultimately precipitates completely from solution between 13 and 14 m LiCl. This aggregation process is dependent on concentration, base composition, and molecular weight of DNA. The sedimentation velocity data taken together with the absorbance spectral data suggest that the aggregation process leading to the formaiton of large structures beings at approximately equal to 9 m. Prior to the onset of aggregation, the circular dichroism (CD) spectra can be adequately fitted by a linear combination of contributions of the B, C, and A forms of DNA (Hanlon, S., Brudno, S., Wu, T. T., and Wolf, B. (1975), Biochemistry 14, 1648). Above 9 m LiCl, both factor analysis and a primitive version of matrix rank order analysis indicate that at least one additional spectral component is required to account for the observed CD spectra above 260 nm. The general shape of this additional component or distortion resembles the psi form of DNA.     

A comparative X-ray diffraction and circular dichroism study of DNA compact particles formed in water-salt solutions, containing poly(ethylene glycol).

Comparative CD and X-ray diffraction studies of DNA compact particules which were obtained in PEG-containing water-salt solutions, have been carried out. Compact particles, formed from native DNA, produce a psi CD spectrum (characterized by a negative band at lambda-270 nm) and a small-angle X-ray diffraction pattern, which shows two reflections: I at 34-40 A and II at 80-90 A (together with its second-order reflection). Compact particules, formed from DNA molecules with partially disordered secondary structure, do not produce the psi CD spectrum and the reflection I, while the reflection II remains unchanged. It is suggested that the spacing of 34-40 A is associated with a side-by-side packing of DNA fragments in "microcrystallization' regions in compact particules and that such "microcrystallization' accounts for the generation of the psi CD spectrum.     

The DnaX-binding subunits delta' and psi are bound to gamma and not tau in the DNA polymerase III holoenzyme

The DnaX complex subassembly of the DNA polymerase III holoenzyme is comprised of the DnaX proteins tau and gamma and the auxiliary subunits delta, delta', chi, and psi, which together load the beta processivity factor onto primed DNA in an ATP-dependent reaction. delta' and psi bind directly to DnaX whereas delta and chi bind to delta' and psi, respectively (Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13348-13357). Until now, it has been unclear which DnaX protein, tau or gamma, in holoenzyme binds the auxiliary subunits delta, delta', chi,and psi. Treatment of purified holoenzyme with the homobifunctional cross-linker bis(sulfosuccinimidyl)suberate produces covalently cross-linked gamma-delta' and gamma-psi complexes identified by Western blot analysis. Immunodetection of cross-linked species with anti-delta' and anti-psi antibodies revealed that no tau-delta' or tau-psi cross-links had formed, suggesting that the delta' and psi subunits reside only on gamma within holoenzyme.     

Cloning, nucleotide sequence and molecular evolution of a rabbit processed metallothionein MT-2 pseudogene

A rabbit metallothionein-2 pseudogene (MT-2 psi) has been isolated from a partial rabbit genomic library. Its unusual sequence shows evidence of complex rearrangements involving recombination and deletion events. There are no intervening sequences, 3' poly A tract or 5' regulatory DNA sequences. The pseudogene is flanked by two sets of direct repeats (CT)3 GT(CT)4 and CTGG(G)CTC. They are most probably the sites of insertion of MT-2 psi in the rabbit genome. In addition, a number of repetitive DNA sequences are observed flanking the MT-2 psi gene. These are features of a processed retrogene.     

Circular dichroism microscopy of compact forms of DNA and chromatin in vivo and in vitro: cholesteric liquid-crystalline phases of DNA and single dinoflagellate nuclei

Two highly condensed structures of DNA have been analyzed in the circular dichroism (CD) microscope: the cholesteric liquid-crystalline phase of DNA and the nucleus of a dinoflagellate (Prorocentrum micans). In both cases, the DNA shows a helical cholesteric organization, but the helical pitch equals about 2500 nm in the first case and 250 nm in the second one. Since the absorption band of DNA is located at 260 nm, the reflection and absorption bands are well separated in the cholesteric phase of DNA and are overlapping in the dinoflagellate nucleus. However, both structures give a very strong negative CD signal at 265 nm. We show that this very strong signal cannot correspond to a Borrmann effect, i.e., to a superposition of the absorption and reflection bands, but is a differential absorption of left versus right circularly polarized light. This anomalous differential absorption is probably due to a significant scattering of light, inside of the structure, which produces a resonance phenomenon in the absorption band of the chromophore. Therefore, for any helical structure containing a chromophore, the apparent CD can be expressed as CD = [(epsilon L - epsilon R)cl] + (psi L - psi R) + (SL - SR) The first term is true absorption and is located in the absorption band of the chromophore, and the last term is true scattering and is observed at the wavelength corresponding to the helical pitch of the structure. The second term (psi L - psi R) corresponds to the anomalous differential absorption observed in dense superhelical structures of DNA. It superimposes to the first term in the absorption band of the chromophore. psi L - psi R is a measure of the perfection of the helical structure and of the density of chromophores in the material. Intercalative dyes [ethidium bromide and meso-tetrakis(4-N-methylpyridyl)porphine (H2TMpyP-4) and its nickel(II) derivative (NiIITMpyP-4)] were inserted in the dinoflagellate chromatin. The CD signal recorded in their absorption band mimics the signal observed in the absorption band of DNA. In both structures, the negative sign of the CD at 265 nm indicates that the twist occurring between DNA. In both structures, the negative sign of the CD at 265 nm indicates that the twist occurring between DNA molecules is left-handed, and we show that this situation is the most frequently encountered in vivo and vitro.     

Kinetic analysis of psi-DNA structure formation induced by histone H1 and its C-terminal domain

In this paper we have studied the kinetics of psi-DNA structure formation induced by H1 and H1 peptides containing the C-terminal domain, namely the CTB peptide, obtained by thrombin digestion, and the CNBS peptide, derived from N-bromosuccinimide treatment of H1. The time course for the formation of the psi structure has been followed by measuring the changes in ellipticity at 270 nm as a function of time under different experimental conditions. In all cases studied here, we have observed the existence of two elementary processes: one fast, the other slow. Kinetic experiments performed with high molecular weight DNA showed that the greater the salt concentration, the higher was the apparent rate of psi structure formation. In complexes formed with sonicated DNA and H1, CNBS and CTB, we observed that the greater the content of the C-terminal domain, the higher was the apparent rate at which the final psi structure was reached. Thus, the presence of increasing amounts of either salt or C-terminal domain facilitates the formation of the psi structure. The molecular basis for these phenomena is discussed. The influence of the order of addition of the different components of the complex on the kinetics of psi structure induction is also studied.