The photoaddition of trimethylpsoralen to Drosophila melanogaster nuclei: a probe for chromatin substructure.

Derivatives of the furocoumarin, psoralen, can penetrate intact cells or nuclei and cross-link opposite strands of the chromosomal DNA under the influence of long wave-length ultraviolet light. The potential of trioxsalen (4,5',8-trimethylpsoralen) as a probe for chromatin structure has been investigated. The DNA in both embryo nuclei and tissue culture cells from Drosophila melanogaster was found to be about 90% protected from trioxsalen binding relative to purified DNA. Digestion of trioxsalen-treated nuclei by micrococcal nuclease and gel electrophoresis of the resulting DNA gave the same type of band pattern that is characteristic of native, untreated nuclei are digestion. Nuclease digestion was therefore used to examine the distribution of bound trioxsalen in the DNA. The resulting DNA fragments were analyzed both by radioactivity measurements and quantitative electron microscopy. The nuclease cleaved intact photoreacted nuclei in such a way that preferential excision of trioxsalen containing regions of the DNA occurred, but, when acting upon purified DNA that contained bount trioxsalen, it attacked the trioxsalen-free regions preferentially. It was thus concluded that trixosalen binds at the sites corresponding to the regular nuclease-sensitive regions of the chromatin in nuclei.     

Properties of Escherichia coli 16S ribosomal ribonucleic acid treated with 4,5',8-trimethylpsoralen and light.

16S rRNA reacted with the furocoumarin 4,5',8-trimethylpsoralen (trioxsalen) and 360-nm light showed a number of chemical and physical differences from untreated RNA. After extensive irradiation, five molecules of trioxsalen were bound per molecule of RNA. The trioxsalen-treated RNA had an altered ultraviolet absorption spectrum and a distinctive fluorescence emission spectrum. The modified RNA was significantly more resistant to T1 ribonuclease digestion than was control RNA. Treated RNA, when mixed with purified ribosomal proteins, was not functional in the in vitro reconstitution of 30S subunits and yielded more slowly sedimenting particles which were inactive in protein synthesis assays. By contrast, 16S rRNA within the 30S subunit structure did not exhibit these changes when reacted with the same dose of trioxsalen and light, suggesting that the ribosomal proteins were effective in protecting the RNA from interaction with the drug.     

Protein-DNA crosslinking in reconstituted nucleohistone, nuclei and whole cells by picosecond UV laser irradiation.

A picosecond UV laser was used to cross-link proteins to DNA in nuclei, whole cells and reconstituted nucleohistone. Irradiation of the nucleohistone resulted in crosslinking 15-20% of bound histones to DNA in a very short time (one or several picosecond pulses), the efficiency of crosslinking to single stranded DNA being higher than to double stranded DNA. All histones as well as high mobility group 1 proteins were identified in the covalently linked protein-DNA complexes upon irradiation of isolated nuclei and whole cells. A method is suggested for isolation of crosslinked material from cells and nuclei in amounts sufficient for further analysis. Experiments with reconstituted nucleohistones showed that upon irradiation at a constant dose the efficiency of crosslinking depended on the intensity of the light, thus suggesting a two-quantum process is involved in the reaction.     

Intranuclear uptake and persistence of biologically active DNA after electroporation of mammalian cells

Radiolabeled or biologically functional DNA molecules were introduced into cells by electroporation in a variety of forms: double stranded circles, linearized double stranded fragments and single stranded circular molecules. Molecules rapidly entered cells after exposure to a high field-strength electric pulse and then redistributed between the cytoplasm and the nucleus. Maximal intranuclear levels approximated 10(4) molecules per cell. Introduced DNA persisted in a biologically active form with a half-life of 15-24 h. There was no evidence for biologically significant alteration of two double stranded gene sequences. Single stranded DNA molecules also retained biological activity.     

Evidence for contamination of origin DNA isolated after an in vivo treatment of mammalian cells with 4,5',8-trimethylpsoralen

We have studied the effect of in vivo treatment with trioxsalen on DNA replication in mammalian cells. In vitro cultured bovine liver cells were exposed to two or four cycles of treatment with 45 microM trioxsalen followed by irradiation with long-wave ultraviolet light. Thymidine incorporation was reduced by about 95% during the first hour after a double treatment. A large proportion of the label was released in alkaline sucrose gradients as a low molecular weight fraction (average length about 500 nucleotides) which was supposed to consist of replication origins containing DNA fragments. From the relative quantities of this DNA obtained at different times of the S phase we concluded that it contains a considerable but not precisely determinable proportion of non-origin DNA. We also find that the fraction is contaminated by a large excess of non-replicating bulk DNA.     

Repair DNA synthesis in heterokaryons during reactivation of chick erythrocytes fused with human diploid fibroblasts or HeLa cells

Suppression of unscheduled DNA synthesis (UDS) after exposure to ultraviolet (UV) light in the human nuclei results when diploid human fibroblasts are fused with chick erythrocytes. The suppression is positively correlated with the number of erythrocyte nuclei in the heterokaryons, with a maximal effect at 36 h after fusion. Evidence is presented that this suppression is due to lowered levels of the enzymes involved in UDS as a result of inhibition of the RNA synthesis by chick components. No suppression of UDS is detected in the human nuclei of the HeLa-chick erythrocyte heterokaryons. In HeLa cells the rate of RNA synthesis is about 10 times higher than the rate in the normal diploid fibroblasts, and the relatively small inhibitory influence of the chick components will therefore not lead to a limitation of the enzymes involved in UDS in the HeLa-chick erythrocyte heterokaryons.     

Kinetics of replicon initiation during S phase of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were synchronized by a thymidine-hydroxyurea block. At different times after release from the block, cells were treated with trioxsalen and long-wavelength ultraviolet light to crosslink DNA in vivo and were labelled with [³H]thymidine for 30 min. This technique permits labelling of only the short nescent DNA fragments initiated between crosslinks. The amount of radioactivity incorporated in these fragments during the labelling period reflects the number of replicon initiation events and allows us to follow the replicon initiation pattern after removing the inhibitor. It was shown that the rate of initiation was high at the beginning of S phase and then steadily decreased.     

Binding of protoberberine alkaloid coralyne with double stranded poly(A): a biophysical study

Recognition of double stranded ribonucleic acid is a critical event in many biological pathways such as trafficking, editing and maturation of mRNA, interferon antiviral response and RNA interference. In the context of probing double stranded RNA binding small molecules, the interaction of the antitumor protoberberine alkaloid coralyne with double stranded poly(A) has been studied by various biophysical techniques. Typical hypochromic and bathochromic shifts in the absorption spectrum and appreciable quenching of the intrinsic fluorescence of coralyne indicated the strong affinity of coralyne to poly(A). The corresponding intrinsic binding constant evaluated from Scatchard analysis was in the order of 10(5) M(-1). The strong binding was further characterized by significant polarization of the alkaloid fluorescence and stabilization of poly(A) helix against thermal strand separation. The binding process was manifested by remarkable perturbation of the intrinsic circular dichroic spectrum of poly(A) with concomitant generation of optical activity in the bound alkaloid molecules that are otherwise achiral. Job plot analysis showed the binding stoichiometry of the interaction process to be two base pairs per alkaloid molecule. The energetics of the strong interaction was studied by isothermal titration and differential scanning calorimetric techniques that suggested the binding to be exothermic and favoured by both negative enthalpy and positive entropy changes. All these results, together with the Stern-Volmer quenching experiment in fluorescence, revealed the molecular details of the intercalation of coralyne into poly(A) duplex leading to its potential use as an agent in gene regulation in eukaryotic cells.     

DNA-binding protein from HeLa cells that binds preferentially to supercoiled DNA damaged by ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene

A DNA-binding protein was partially purified from extracts of HeLa cells by high-speed centrifugation and chromatography on DEAE-cellulose, phosphocellulose and ultraviolet light-irradiated DNA-cellulose columns. It eluted from the phosphocellulose column with 0.375 M potassium phosphate and from the ultraviolet light-irradiated DNA-cellulose column between 0.5 M and 1 M NaCl. The protein binds preferentially to supercoiled PM2 DNA treated with ultraviolet light or N-acetoxy-N-acetyl-2-aminofluorene, as compared to native supercoiled PM2 DNA. The binding is non-cooperative. Nicked or linear forms of PM2 DNA (damaged or untreated) are not efficient substrates, indicating a requirement of DNA supercoiling for DNA binding. The sedimentation coefficient of the protein estimated by glycerol gradient centrifugation is 2.0–2.5 S, corresponding to a molecular weight of about 20 000–25 000 if the protein is spherical. The binding to DNA irradiated with ultraviolet light or treated with acetoxyacetylaminofluorene is optimal at around 100–200 mM NaCl and is relatively independent of temperature and pH. MgCl₂ and MnCl₂ at concentrations between 1 and 5 mM do not markedly affect the binding, but it is inhibited by sucrose, ATP and caffeine. The biological significance of the DNA-binding protein remains to be determined. It does not possess significant glycosylase, endonuclease or exonuclease activities. The dissociation equilibrium constant for the binding reaction of the protein to the ultraviolet light or acetoxyacetylaminofluorene-induced binding sites on DNA is estimated to be 4·10^?11 M. There are at least 1·10⁵ DNA-binding protein molecules/HeLa cell.     

Small molecule intercalation with double stranded DNA: implications for normal gene regulation and for predicting the biological efficacy and genotoxicity of drugs and other chemicals

The binding of small molecules to double stranded DNA including intercalation between base pairs has been a topic of research for over 40 years. For the most part, however, intercalation has been of marginal interest given the prevailing notion that binding of small molecules to protein receptors is largely responsible for governing biological function. This picture is now changing with the discovery of nuclear enzymes, e.g. topoisomerases that modulate intercalation of various compounds including certain antitumor drugs and genotoxins. While intercalators are classically flat, aromatic structures that can easily insert between base pairs, our laboratories reported in 1977 that a number of biologically active compounds with greater molecular thickness, e.g. steroid hormones, could fit stereospecifically between base pairs. The hypothesis was advanced that intercalation was a salient feature of the action of gene regulatory molecules. Two parallel lines of research were pursued: (1) development of technology to employ intercalation in the design of safe and effective chemicals, e.g. pharmaceuticals, nutraceuticals, agricultural chemicals; (2) exploration of intercalation in the mode of action of nuclear receptor proteins. Computer modeling demonstrated that degree of fit of certain small molecules into DNA intercalation sites correlated with degree of biological activity but not with strength of receptor binding. These findings led to computational tools including pharmacophores and search engines to design new drug candidates by predicting desirable and undesirable activities. The specific sequences in DNA into which ligands best intercalated were later found in the consensus sequences of genes activated by nuclear receptors implying intercalation was central to their mode of action. Recently, the orientation of ligands bound to nuclear receptors was found to match closely the spatial locations of ligands derived from intercalation into unwound gene sequences suggesting that nuclear receptors may be guiding ligands to DNA with remarkable precision. Based upon multiple lines of experimental evidence, we suggest that intercalation in double stranded DNA is a ubiquitous, natural process and a salient feature of the regulation of genes. If double stranded DNA is proven to be the ultimate target of genomic drug action, intercalation will emerge as a cornerstone of the future discovery of safe and effective pharmaceuticals.     

Fluorescence of proflavine--DNA complexes: heterogeneity of binding sites

Measurements of the relative quantum yield of fluorescence of proflavine bound to DNA as a function of the number of bound dyes per nucleotide and the ionic strength allow the determination of the binding constants and respective number of the two types of sites previously postulated. It is demonstrated that 2–3% of the base pairs form sites where the dye is strongly bound and fluoresces normally while in the other set of sites the binding constant is 3–4 times weaker and the fluorescence completely quenched. Comparison with complexes of Pro with double stranded polynucleotides poly (A + U), poly (I + C), poly(G + C), confirm that the strong binding sites correspond to A-T-rich regions of the DNA while the quenched sites seem to require the presence of a neighboring guanine. The role of charge transfer in quenching of fluorescence and mutagnic action is considered. An original method for the determination of free dye and bound dye, based upon the use of an external quencher is described in the Appendix.     

Identification of the short dispersed repetitive DNA sequences isolated from the zones of initiation of DNA synthesis in human cells as Alu-elements

DNA of Xeroderma pigmentosum cells was crosslinked in vivo with trioxsalen and long wave length ultraviolet light and the cells were cultured in the presence of labelled thymidine for one hour. The nascent DNA chains synthesized during this period and containing the DNA replication origins were isolated from the high molecular weight chromosomal DNA by an alkaline sucrose density gradient centrifugation. They were 5-10-fold enriched in short dispersed repetitive sequences identified by dot-blot hybridization to BLUR 8 plasmid as members of the human Alu-family.     

Evidence for contamination of origin DNA isolated after an in vivo treatment of mammalian cells with 4,5′,8-trimethylpsoralen

We have studied the effect of in vivo treatment with trioxsalen on DNA replication in mammalian cells. In vitro cultured bovine liver cells were exposed to two or four cycles of treatment with 45 μM trioxsalen followed by irradiation with long-wave ultraviolet light. Thymidine incorporation was reduced by about 95% during the first hour after a double treatment. A large proportion of the label was released in alkaline sucrose gradients as a low molecular weight fraction (average length about 500 nucleotides) which was supposed to consist of replication origins containing DNA fragments. From the relative quantities of this DNA obtained at different times of the S phase we concluded that it contains a considerable but not precisely determinable proportion of non-origin DNA. We also find that the fraction is contaminated by a large excess of non-replicating bulk DNA.     

4.5S RNA is encoded by hundreds of tandemly linked genes, has a short half-life, and is hydrogen bonded in vivo to poly(A)-terminated RNAs in the cytoplasm of cultured mouse cells.

4.5S RNA is a group of RNAs 90 to 94 nucleotides long (length polymorphism due to a varying number of UMP residues at the 3' end) that form hydrogen bonds with poly(A)-terminated RNAs isolated from mouse, hamster, or rat cells (W. R. Jelinek and L. Leinwand, Cell 15:205-214, 1978; F. Harada, N. Kato, and H.-O. Hoshino, Nucleic Acids Res. 7:909-917, 1979). We have cloned a gene that encodes the 4.5S RNA. It is repeated 850 (sigma = 54) times per haploid mouse genome and 690 (sigma = 59) times per haploid rat genome. Most, if not all, of the repeats in both species are arrayed in tandem. The repeat unit is 4,245 base pairs long in mouse DNA (the complete base sequence of one repeat unit is presented) and approximately 5,300 base pairs in rat DNA. This accounts for approximately 3 X 10(6) base pairs of genomic DNA in each species, or 0.1% of the genome. Cultured murine erythroleukemia cells contain 13,000 molecules per cell of the 4.5S RNA, which can be labeled to equilibrium in 90 min by [3H]uridine added to the culture medium. The 4.5S RNA, therefore, has a short half-life. The 4.5S RNA can be cross-linked in vivo by 4'-aminomethyl-4,5',8-trimethylpsoralen to murine erythroleukemia cell poly(A)-terminated cytoplasmic RNA contained in ribonucleoprotein particles.     

Proteins tightly bound to HeLa cell DNA at nuclear matrix attachment sites.

DNA-protein complexes have been isolated from HeLa cell nuclei and nuclear matrix preparations. Two proteins, 55 and 66 kilodaltons in size, remain bound to HeLa DNA after treatment at 80 degrees C in 2% sodium dodecyl sulfate and purification by exclusion chromatography on Sepharose 2B-CL in the presence of 0.3% sodium dodecyl sulfate. These proteins appear to be tightly bound but not covalently linked to the DNA, and they are distributed over the DNA with an average spacing of 40 kilobase pairs. This spacing distribution remains essentially constant throughout the cell cycle. The proteins are bound to the residual 2% of HeLa cell DNA which remains attached to the nuclear matrix after extensive nuclease digestion, a condition which reduces the average size of the DNA to approximately 150 base pairs. Our results suggest that these tightly bound proteins are involved in anchoring cellular DNA to the nuclear matrix. These tightly bound proteins are identical by partial peptide mapping to proteins found tightly bound to the DNA of mammalian, plant, and bacterial cells (D. Werner and C. Petzelt, J. Mol. Biol. 150:297-302, 1981), implying that these proteins are involved in the organization of chromosomal domains and are highly conserved in both procaryotic and eucaryotic cells.     

A DNA helicase from human cells.

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.