Giant proteins that move DNA: bullies of the genomic playground

As genetic material DNA is wonderful, but as a macromolecule it is unruly, voluminous and fragile. Without the action of DNA replicases, topoisomerases, helicases, translocases and recombinases, the genome would collapse into a topologically entangled random coil that would be useless to the cell. We discuss the organization, movement and energetics of these proteins that are crucial to the preservation of a molecule that has such beautiful biological but challenging physical properties.     

Inhibition of Escherichia coli K12 by short-chain organic acids: lack of evidence for induction of the SOS response

Sublethal concentrations of formic acid (10 mmol/l) and propionic acid (5 mmol/l) at pH 5.0 preferentially inhibit DNA synthesis and stop cell multiplication in the absence of a corresponding cessation in the increase of culture turbidity. The possibility that the acids induce the SOS response by starving cells of thymine or by causing physical damage to the DNA molecule has now been investigated. Accumulation of thymine into the cytoplasm of whole cells was not inhibited by either acid. Mutants defective in excision repair ( uvrA6 ), recombination repair ( recA56 ) and polymerase activity ( polA1 ) were not more sensitive to the acids than their isogenic parent. No significant increase in cell length was observed from measurements of transmission electron microscope images of acid-treated cells. It is concluded, therefore, that sublethal concentrations of formic and propionic acid inhibit DNA synthesis without physically damaging DNA molecule, or starving the cell of essential thymine or otherwise inducing an SOS response.     

Interfacial spin trapping in model membrane systems

The nature of mitochondrial DNA replication during the synchronous cell cycle in the yeast, $\text{Saccharomyces}\text{cerevisiae}$ has been investigated by examining the rate of labeled DNA precursor incorporation into specific segments of the mitochondrial genome at defined points during synchronous growth. The movement of label uptake from one area of the DNA to another at different times during the synchronous cell cycle indicates mitochondrial DNA replication to be a synchronous process during this time with most or all molecules at the same point in replication at any given time during the cell cycle.     

Bacterial translocation motors investigated by single molecule techniques

Translocation of DNA and protein fibers through narrow constrictions is a ubiquitous and crucial activity of bacterial cells. Bacteria use specialized machines to support macromolecular movement. A very important step toward a mechanistic understanding of these translocation machines is the characterization of their physical properties at the single molecule level. Recently, four bacterial transport processes have been characterized by nanomanipulation at the single molecule level, DNA translocation by FtsK and SpoIIIE, DNA import during transformation, and the related process of a type IV pilus retraction. With all four processes, the translocation rates, processivity, and stalling forces were remarkably high as compared with single molecule experiments with other molecular motors. Although substrates of all four processes proceed along a preferential direction of translocation, directionality has been shown to be controlled by distinct mechanisms.     

New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome.

A set of plasmid cloning vectors has been constructed, allowing the integration of any DNA fragment into the bacteriophage lambda attachment site attB of the Escherichia coli chromosome. The system is based upon two components: (i) a number of cloning vectors containing the lambda attachment site attP and (ii) a helper plasmid, bearing the lambda int gene, transcribed from the lambda PR promoter under the control of the temperature-sensitive repressor cI857. The DNA fragment of interest is cloned into the multicloning site of one of the attP-harboring plasmids. Subsequently, the origin of the plasmid, located on a cloning cassette, is cut out and the DNA becomes newly ligated, resulting in a circular DNA molecule without replication ability. The strain of choice, containing the int gene carrying helper plasmid, is transformed with this DNA molecule and incubated at 42 degrees C to induce int gene expression. Additionally, the temperature shift leads to the loss of the helper plasmid after a few cell generations, because the replication ability of its replicon is blocked at 42 degrees C. These vectors have been successfully used for integration of several promoter-lacZ fusions into the chromosome. The ratio between integration due to homologous recombination and Int protein-mediated integration has been determined.     

Features of the geometry of a supercoiled DNA molecule

The features of the geometry of a supercoiled DNA molecule are considered. A model of supercoiled structure of DNA taking into account its natural helical structure has been constructed. Force factors arising in DNA has been calculated depending on winding angle.     

Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.     

Inhibition of Escherichia coli K12 by short-chain organic acids: lack of evidence for induction of the SOS response

Sublethal concentrations of formic acid (10 mmol/l) and propionic acid (5 mmol/l) at pH 5.0 preferentially inhibit DNA synthesis and stop cell multiplication in the absence of a corresponding cessation in the increase of culture turbidity. The possibility that the acids induce the SOS response by starving cells of thymine or by causing physical damage to the DNA molecule has now been investigated. Accumulation of thymine into the cytoplasm of whole cells was not inhibited by either acid. Mutants defective in excision repair (uvr A6), recombination repair (rec A56) and polymerase activity (pol A1) were not more sensitive to the acids than their isogenic parent. No significant increase in cell length was observed from measurements of transmission electron microscope images of acid-treated cells. It is concluded, therefore, that sublethal concentrations of formic and propionic acid inhibit DNA synthesis without physically damaging DNA molecule, or starving the cell of essential thymine or otherwise inducing an SOS response.     

The mitotic cycle and DNA repair in UV-irradiated murine neuroblastoma cells]

A correlation has been shown between a reduced rate of movement of UV-irradiated neuroblastoma cells from G1 into S phase, an essential increase of cells in S phase while progressing through the cell cycle, and a defect in free DNA synthesis on a damaged template. These indices may reflect one and the same cell response to the UV light.     

Expression of Semliki Forest virus proteins from cloned complementary DNA. I. The fusion activity of the spike glycoprotein

A complementary (cDNA) molecule encoding the structural proteins of Semliki Forest virus (SFV) has been inserted into a Simian virus 40- derived eucaryotic expression vector lacking introns. Introduction of the recombinant DNA into nuclei of baby hamster kidney cells results in the synthesis of authentic SFV membrane glycoproteins E1 and E2. The glycoproteins are both transported to the cell surface and induce cell- cell fusion after a brief treatment of the cells with low pH medium. The pH dependence of the fusion reaction was the same as that induced by virus particles (White, J., J. Kartenbeck, and A. Helenius, 1980, J. Cell Biol., 89:674-679). Transfection of cells with another recombinant DNA molecule in which the SFV cDNA is engineered into the same expression vector including an intron has been shown before to result in the expression of only the E2 protein on the cell surface, whereas the E1 protein is trapped in the rough endoplasmic reticulum (Kondor- Koch, C., H. Riedel, K. Soderberg, and H. Garoff, 1982, Proc. Natl. Acad. Sci. USA, 79:4525-4529). Such cells do not exhibit pH-dependent polykaryon formation, suggesting that the E1 protein is necessary for fusion activity. Immunoblotting experiments show that the RER-trapped E1 protein expressed from the DNA construction with an intron has a smaller apparent molecular weight than authentic E1, and that is has lost its amphipathic characteristics.     

Identification and characterization of an inhibitor specific to bacterial NAD+-dependent DNA ligases

DNA ligases are the enzymes essential for DNA replication, repair and recombination in all organisms. The bacterial DNA ligases involved in DNA replication require NAD(+) for activity, but eukaryotic and viral DNA ligases require ATP. Because of their essential nature, unique structures and widespread existence in nature, bacterial DNA ligases represent a class of valuable targets for identifying novel and selective antibacterial agents. In this study, we cloned and expressed the ligA gene from Streptococcus pneumoniae, and characterized this ligA-encoded NAD(+)-dependent DNA ligase. We then screened small molecule chemical libraries using a biochemical assay and identified a new small molecule with a structure of 2,4-diamino-7-dimethylamino-pyrimido[4,5-d]pyrimidine. We show that this small molecule is a specific inhibitor of bacterial NAD(+)-dependent DNA ligases. Biochemical studies show that this molecule inhibits NAD(+)-dependent DNA ligases, but not ATP-dependent enzymes. The molecule inhibits NAD(+)-dependent DNA ligases competitively with respect to NAD(+) and specifically inhibits enzyme adenylation, but not DNA adenylation or ligation. Labeling studies establish that this molecule inhibits the incorporation of thymidine into DNA and that overexpression of DNA ligase in the cell abolishes this inhibition. Finally, microbiological studies show that this molecule exhibits a broad spectrum of antibacterial activity. Together, this study shows that this small molecule inhibitor identified is specific to bacterial NAD(+)-dependent DNA ligases, exhibits a broad spectrum of antibacterial activities, and has the potential to be developed into an antibacterial agent.     

Receptor-mediated photo-cytotoxicity: synthesis of a photoactivatable psoralen derivative conjugated to insulin

4'-Aminomethyl-4,5',8-trimethylpsoralen has been chemically conjugated to insulin using a carbodiimide derivative. The psoralen moiety retains its photochemical reactivity as evidenced by its ability to crosslink DNA after exposure to long wavelength ultraviolet light (UVA, 320-400 nm). This chimeric molecule has been used to selectively kill a population of lymphocytes whose expression of insulin receptors has been stimulated with phytohemagglutinin. Insulin carries the psoralen into the cell via receptor-mediated endocytosis, where it is subsequently activated by exposure to UVA light. The UVA induced activity of AMT-insulin can be blocked by the presence of native insulin. The viability of unstimulated lymphocytes was not affected by AMT-insulin and UVA light. The hybrid insulin-psoralen molecule may be a prototype for a family of phototoxic drugs which can be selectively delivered to subsets of lymphocytes.     

A measurement of the fraction of chloroplast DNA transcribed in Euglena

The fraction of the chloroplast DNA transcribed in the single celled alga $\text{Euglena}$ has been determined by RNA-DNA hybridization. A vast excess of total cell RNA from cells which were rapidly dividing in the light was hybridized in liquid to [¹²⁵I] — chloroplast DNA, and the resulting duplexes separated on hydroxyapatite columns. The contribution of DNA-DNA duplex formation was determined separately and was used to calculate that portion of the duplex which was actually a RNA-DNA hybrid. Sixteen percent of the single stranded chloroplast DNA forms a duplex with this RNA suggesting that 32 percent of the double stranded DNA molecule is being transcribed into RNA under these conditions of cell growth.     

Regulated replication of DNA microinjected into eggs of Xenopus laevis

Purified circular DNA of SV40 or polyoma virus has been injected into unfertilized eggs of Xenopus laevis. Injected DNA initiates and completes multiple rounds of semiconservative replication while observing cellular regulatory signals. Thus replication initiation of double-stranded templates is induced after the oocyte is matured in vitro by progesterone. Only one round of replication of injected DNA is observed in a single cell cycle. When protein synthesis is inhibited unreplicated molecules continue to initiate replication at an undiminished rate, but reinitiation on previously replicated molecules is completely and selectively abolished. The DNA sequence requirements for the replication of injected DNA have been investigated. A variety of procaryotic DNA molecules and circularized fragments of SV40 or polyoma DNA replicate, regardless of whether they contain the viral origin of DNA replication. These results suggest that a specialized DNA sequence is not essential for the initiation of semiconservative DNA replication in the Xenopus embryo, nor is a specialized sequence essential for the mechanism which prevents reinitiation on a molecule which has already replicated within a cell cycle. The possibility is discussed that viral origins of replication are not valid models for the eucaryotic chromosome but are adaptations for uncoupling viral replication from the mechanism which prevents reinitiation within a cell cycle.     

Two-dimensional (2D) DNA crystals assembled from two DNA strands

Sequence symmetry has been used to simplify the design of a DNA double-crossover (DX) molecule. The resulting DX molecule can self-assemble into two-dimensional (2D) crystalline arrays, but only requires two instead of otherwise four different DNA strands.     

Fibroblast Growth Factor Receptor-induced Phosphorylation of EphrinB1 Modulates Its Interaction with Dishevelled

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. The transmembrane ephrinB1 protein is a bidirectional signaling molecule that signals through its cytoplasmic domain to promote cellular movements into the eye field, whereas activation of the fibroblast growth factor receptor (FGFR) represses these movements and retinal fate. In Xenopus embryos, ephrinB1 plays a role in retinal progenitor cell movement into the eye field through an interaction with the scaffold protein Dishevelled (Dsh). However, the mechanism by which the FGFR may regulate this cell movement is unknown. Here, we present evidence that FGFR-induced repression of retinal fate is dependent upon phosphorylation within the intracellular domain of ephrinB1. We demonstrate that phosphorylation of tyrosines 324 and 325 disrupts the ephrinB1/Dsh interaction, thus modulating retinal progenitor movement that is dependent on the planar cell polarity pathway. These results provide mechanistic insight into how fibroblast growth factor signaling modulates ephrinB1 control of retinal progenitor movement within the eye field.     

Inversion and circularization of the varicella-zoster virus genome.

The genome of varicella-zoster virus (VZV) is a linear, double-stranded molecule of DNA composed of a long (L) region covalently linked to a short (S) region. The S region is capable of inverting relative to a fixed orientation of the L region, giving rise to two equimolar populations. We have investigated other forms of the VZV genome which are present in infected cells and packaged into nucleocapsids. That a small proportion of nucleocapsid DNA molecules also possess inverted L regions has been verified by the identification of submolar restriction fragments corresponding to novel joints and novel ends generated by such an inversion. The presence of circular molecules has been investigated by agarose gel electrophoresis. Bands corresponding to circular forms were present in small amounts in both VZV-infected cell DNA and nucleocapsid DNA. Southern blot analysis verified that these bands contained VZV sequences. We therefore conclude that the VZV genome may occasionally contain an inverted L region or exist in a circular configuration.     

Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein

Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread.