Molecular aspects on the interaction of sanguinarine with B-form, Z-form and HL-form DNA structures

The interaction of sanguinarine with right-handed (B-form), left-handed (Z-form) and left-handed (HL-form) structures of poly(dG-dC).poly(dG-dC) has been investigated by measuring the circular dichroism (CD) and UV-absorption spectral analysis. Sanguinarine binds strongly to the B-form DNA and does not bind to Z-form or HL-form, but it converts the Z-form and the HL-form back to the bound right handed form as evidenced from CD spectroscopy. Sanguinarine inhibits the rate of B to Z transition under ionic conditions that otherwise favour the left-handed conformation of the polynucleotides. UV absorption kinetic studies show that the Z-form reverses back to B-form to B-form on binding to sanguinarine. Binding isotherms obtained from spectrophotometric data show that sanguinarine binds strongly to the B-form polymer in a non-cooperative manner, in sharp contrast to the highly cooperative interaction under Z-form and HL-form polynucleotides. These studies reveal that the alternating GC sequence undergoes defined conformational changes and interacts with sanguinarine which may be an important aspect in understanding its extensive biological activities.     

Excision of apurinic sites from DNA with enzymes isolated from rat-liver chromatin

Apurinic sites were excised from phi X174 RF DNA with two enzymes isolated from rat liver chromatin: an apurinic/apyrimidinic endodeoxyribonuclease and a 5'-3'-exonuclease; the resulting gap was filled with DNA polymerase beta also prepared from rat liver chromatin and the repair was fully terminated with T4 ligase.     

Reconstitution of nativelike nuclear acceptor sites of the avian oviduct progesterone receptor: evidence for involvement of specific chromatin proteins and specific DNA sequences

A specific fraction of avian oviduct chromosomal proteins can be reannealed to pure avian DNA to reconstitute nativelike specific nuclear binding sites (acceptor sites) for the oviduct progesterone receptor (PR). These specific nuclear binding sites represent the difference between the binding to the reconstituted NAP and that to pure DNA. The specific fraction of chromatin protein which contains the acceptor activity, fraction CP-3, is very tightly bound to hen DNA in a complex termed nucleoacidic protein (NAP). Removal of the CP-3 fraction from NAP results in a loss of specific PR binding sites. Resins containing chromatin adsorbed to hydroxylapatite are used as a rapid method to isolate the CP-3 fraction. Reconstitution of the CP-3 fraction to DNA by the described method involving a regressing gradient of 6-0 M guanidine hydrochloride (Gdn-HCl) results in a reconstituted NAP which displays specific PR binding sites identical with those in native (undissociated) NAP and whole chromatin. Optimal conditions and potential problems for reconstituting these nucleoproteins are described. Only partially purified receptor preparations were used in these cell-free binding analyses since they have been shown to bind with similar properties and patterns as the nuclear binding in vivo. Therefore, the binding of PR to the reconstituted NAPs was demonstrated to be receptor dependent, saturable, and of high affinity. Further, the pattern of binding to the reconstituted sites mimics those which are observed in vivo. Thus, nonfunctional receptors that cannot translocate and bind to the nuclear acceptor sites in vivo also failed to bind to the acceptor sites on the reconstituted NAPs generated by the acceptor proteins. In contrast, the binding to pure DNA does not reflect these receptor differences in receptor bindings. Specific binding of PR to reconstituted NAP can be reversed by again removing the protein fraction. Moreover, the specific binding can be destroyed by proteases and protected by protease inhibitors, indicating that acceptor activity is proteinaceous in nature. The reconstitution of the activity is both a concentration-dependent and time-dependent process. During the reconstitution, acceptor activity appears to reconstitute on the DNA when the Gdn-HCl concentration reaches 2.0 M. By use of the reconstitution method as an assay for acceptor activity, the activity in the CP-3 fraction was shown by molecular sieve chromatography to elute in a relatively broad molecular weight range between 13 000 and 25 000. The activity also focuses in isoelectric focusing resins with apparent pI's of 5.2 and 6.4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster.

Three new proteins which selectively bind to UV-damaged DNA were identified and purified to near homogeneity from UV-irradiated Drosophila melanogaster embryos through several column chromatographies. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrated that it is a monomer protein which is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrated that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. These findings are discussed in relation to two recently described [Todo and Ryo (1991) Mutat. Res., 273, 85-93; Todo et al. (1993) Nature, 361, 371-374] DNA-binding factors from Drosophila cell extracts. A possible role for these DNA-binding proteins in lesion recognition and DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products is discussed.     

Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold

Data are presented for sequence-specific chromatin-loop organization in histone-depleted nuclei from Drosophila melanogaster Kc cells. We find one loop for each of the tandemly repeated histone gene clusters. The attachment site is localized in the A + T rich H1-H3 spacer on a 657 bp fragment. In the cluster of the hsp70 heat-shock genes, in both control and heat-shocked cells, we find two attachment sites in close proximity upstream of regulatory elements. The transcribed sequences are not associated with the nuclear scaffold in control or in heat-shocked cells. A family of attachment sites related by hybridization to those of the hsp70 genes was discovered.     

How proteins search for their specific sites on DNA: the role of DNA conformation

It is known since the early days of molecular biology that proteins locate their specific targets on DNA up to two orders-of-magnitude faster than the Smoluchowski three-dimensional diffusion rate. An accepted explanation of this fact is that proteins are nonspecifically adsorbed on DNA, and sliding along DNA provides for the faster one-dimensional search. Surprisingly, the role of DNA conformation was never considered in this context. In this article, we explicitly address the relative role of three-dimensional diffusion and one-dimensional sliding along coiled or globular DNA and the possibility of correlated readsorption of desorbed proteins. We have identified a wealth of new different scaling regimes. We also found the maximal possible acceleration of the reaction due to sliding. We found that the maximum on the rate-versus-ionic strength curve is asymmetric, and that sliding can lead not only to acceleration, but also in some regimes to dramatic deceleration of the reaction.     

Analysis of highly purified satellite DNA containing chromatin from the mouse.

A purification scheme for satellite DNA containing chromatin from mouse liver has been developed. It is based on the highly condensed state of the satellite chromatin and also takes advantage of its resistance to digestion by certain restriction nucleases. Nuclei are first treated with micrococcal nuclease and the satellite chromatin enriched 3-5 fold by extraction of the digested nuclei under appropriate conditions. Further purification is achieved by digestion of the chromatin with a restriction nuclease that leaves satellite DNA largely intact but degrades non-satellite DNA extensively. In subsequent sucrose gradient centrifugation the rapidly sedimenting chromatin contains more than 70% satellite DNA. This material has the same histone composition as bulk chromatin. No significant differences were detected in an analysis of minor histone variants. Nonhistone proteins are present only in very low amounts in the satellite chromatin fraction, notably the HMG proteins are strongly depleted.     

Selective prediction of interaction sites in protein structures with THEMATICS

Background  

Methods are now available for the prediction of interaction sites in protein 3D structures. While many of these methods report high success rates for site prediction, often these predictions are not very selective and have low precision. Precision in site prediction is addressed using Theoretical Microscopic Titration Curves (THEMATICS), a simple computational method for the identification of active sites in enzymes. Recall and precision are measured and compared with other methods for the prediction of catalytic sites.     

Mechanical detection of interaction of small specific ligands with proteins and DNA in cross-linked samples.

Cross-linked crystalline and amorphous films of different proteins and cross-linked DNA gels were found to change their mechanical properties when soaked in solutions of specific ligands at nearly physiological concentrations. This chemomechanical effect may be used to rapidly (within a few minutes) detect the ability of macromolecules to bind small (less than 1 kDa) ligand molecules, to measure concentrations of ligands (higher than 10 nM), and to estimate binding constants (lower than 10(7) M-1). Only 0.1-1 mg of protein or DNA is needed to prepare more than 10 samples sufficient for a large number of tests, provided binding is reversible. The method is recommended for rapid primary screening in search of new drugs, in biochemical studies, and as a basis for designing biosensors and other analytical instruments.     

The interaction of cisplatin and analogues with DNA in reconstituted chromatin

The influence of chromatin structure on cis-diamminedichloroplatinum(II) (cisplatin) DNA damage was investigated in a reconstituted nucleosome system. Nucleosomes were reconstituted on the somatic 5S rRNA gene from Xenopus borealis using the octamer transfer method of reconstitution. Footprinting techniques, utilising bleomycin and DNase I as the damaging agents, were employed to establish the precise location of positioned nucleosomes with respect to the DNA sequence. Reconstituted nucleosomal DNA was treated with cisplatin and drug-induced DNA adduct formation was quantitatively analysed with a polymerase stop assay using Taq DNA polymerase. A densitometric comparison of the relative damage band intensities between purified and reconstituted DNA revealed regions of relative protection corresponding to the sites of the positioned nucleosome cores. This indicated that the preferred site of cisplatin DNA binding was in the linker region of the nucleosome. Statistical analysis showed significant protection from cisplatin DNA damage in the core region of the nucleosome. Three cisplatin analogues were also investigated in this reconstituted nucleosome system. These analogues, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin), cis-dichlorobis(cyclohexylamine)platinum(II) (cis-[PtCl(2)(C(6)H(11)NH(2))(2)]) and dichloro(N-[3-[(2-aminoethyl)-amino]propyl]acridine-4-carboxamide)platinum(II) (ac-PtenCl(2)(n3)), were also found to target the linker region of the nucleosome. The latter DNA-targeted acridine-platinum complex gave rise to the most predominant footprints of all the Pt compounds tested.     

Maximum-likelihood estimation of phylogeny from DNA sequences when substitution rates differ over sites

Felsenstein's maximum-likelihood approach for inferring phylogeny from DNA sequences assumes that the rate of nucleotide substitution is constant over different nucleotide sites. This assumption is sometimes unrealistic, as has been revealed by analysis of real sequence data. In the present paper Felsenstein's method is extended to the case where substitution rates over sites are described by the gamma distribution. A numerical example is presented to show that the method fits the data better than do previous models.      

Isolation of a chromatin fraction from calf endometrium highly enriched in estradiol binding sites.

The intranuclear distribution of [3H]-estradiol binding sites was studied in highly purified nuclei isolated from calf endometrial tissue pre-incubated with the labeled hormone. The major part (approximately 85%) of the receptor bound estradiol was found associated with the extranucleolar chromatin; only a negligible amount of [3H]-estradiol (approximately 8%) sedimented with the nucleolar fraction. [3H]-estradiol labeled chromatin was then fragmented by sonication and fractionated by sucrose density gradient sedimentation under different conditions of centrifugation. The vast majority of the [3H]-estradiol was invariably found to be associated with a fast sedimenting fraction which contained only 5 to 10% of the nuclear DNA. The concentration of estradiol receptors (per weight of DNA) in this fraction was 25- to 50-fold higher than that found in the slow sedimenting major chromatin component. Chemical analysis showed this fraction to have a high protein/DNA ratio but no phospholipids were detected.     

Localization of chromatin proteins within DNA grooves by methylation of chromatin with dimethyl sulphate

The use of the comparative modification with ³H-dimethyl sulphate (DMS) of free DNA and DNA in different complexes is proposed to evaluate the shielding of the minor and major grooves of the DNA double helix and to determine the presence of single-stranded DNA in the complexes.Glucosyl groups in DNA of T6 phage protect, as expected, the major groove, and actinomycin d in its complex with DNA shields the minor groove against methylation with DMS.The data obtained suggest that histones and protamine in reconstituted nucleohistone and nucleoprotamine are allocated within partly the major groove leaving the minor groove open, while polylysine does not seem to be buried within either of the grooves, and cations of cetyltrimethylammonium lie within the minor groove of DNA.     

Changes in the interaction of DNA with chromatin and nuclear matrix proteins during digestion of nuclei with restrictases and nuclease Bal 31

Earlier experiments with the use of nucleoprotein-celite chromatography revealed that DNA is bound to a replicative complex localized in the nuclear matrix by a topologically tight bond. Induction of site-specific DNA breaks by restriction nucleases in isolated nuclei of proliferating cells causes a gradual concentration-dependent liberation of DNA from the tight binding to the nuclear matrix. The DNA involved in the tight interaction with matrix proteins is especially sensitive to digestion by Sau 3A1, EcoRI, PstI, BCNI and Bam HI restrictases. One-strand DNA-specific nuclease Bal 31 also destroys the tight DNA-matrix bond. The tightness of DNA-protein bonds in chromatin particles formed after the digestion of nuclei with restrictases is dependent on the particle size. The data are summarized in a model of a topological DNA-matrix bond.     

Highly specific prediction of phosphorylation sites in proteins

SUMMARY: The prediction of significant short functional protein sequences has inherent problems. In predicting phosphorylation sites, problems came from the shortness of phosphorylation sites, the difficulties in maintaining many different predefined models of binding sites, and the difficulties of obtaining highly sensitive predictions and of obtaining predictions with a constant sensitivity and specificity. The algorithm presented in this paper overcomes these problems. The proposed algorithm PHOSITE is based on the case-based sequence analysis. This enables the prediction of phosphorylation sites with constant specificity and sensitivity. Furthermore, this method leads not only to the prediction of phosphorylation sites in general but also predicts the most probable type of kinase involved. AVAILABILITY: The tool PHOSITE implementing the presented method can be evaluated under the website http://www.phosite.com.     

S-N transition of the saccharide ring in B-form DNA

A conformational transition of a single deoxyribose was analyzed in B-form trimers dA3:dT3 and dG3:dC3, both in the purine and pyrimidine chains. The main results were obtained for the duplexes with frozen ends, which could be extended by regular double helixes. The geometry of the central sugar ring in the duplexes may strongly deviate from the regular conformation. When deoxyribose changed its conformation in the central pyrimidine, the energy increase was proved to be less significant in comparison with that for purine. In the case of Thy, a decrease in pseudorotation angle P from 140 to 80 degrees causes the energy increase of 0.5 kcal/mol only, the barrier being 1.2 kcal/mol. The energy profile for Cyt has several local minima. The results of calculations were compared with numerous experimental data, they help to explain some NMR data. A perturbation of the duplex AAA:TTT structure caused by the thymine sugar ring transition, produces 5 degrees bend of the DNA axis directed toward adenines. We also investigated the influence of such conformational disturbance on the neighbouring base pairs, in particular the transition in the trimers with unfrozen ends.