Ralph R. Meyer

RNA synthesis by isolated mammalian mitochondria and nuclei: Effects of ethidium bromide and acriflavin

Abstract The effects of ethidium bromide and acriflavin on RNA synthesis by isolated rat liver mitochondria and nuclei were studied. Mitochondrial RNA synthesis was found to be much more sensitive to these dyes than is nuclear RNA synthesis. At low concentrations both drugs actually stimulated RNA synthesis by isolated nuclei, but inhibited at higher concentrations. Thus, it was suggested that stimulation may be due to removal of repressors since RNA synthesis using solubilized rat liver RNA polymerase and naked DNA as template failed to show such stimulation and was inhibited by much lower concentrations of dyes. Acriflavin was less effective as an inhibitor than ethidium bromide at any given concentration. Results with calf liver organelles were identical to those obtained with rat liver.

DNA biosynthesis in nuclei: I. Characterization of DNA synthesis by isolated rat liver nuclei using endogenous DNA as primer

Abstract 1. A DNA biosynthetic system consisting of isolated nuclei using endogenous DNA as primer has been characterized. The nuclei are capable of incorporating [ 3 H] dTTP into DNA linearly for 2 h, and incorporation is proportional to protein concentration up to 2 mg/ml. 2. Preincubation at 37 °C greatly enhances incorporation, but heating for 4 min at 65 °C destroys activity. The pH optimum is 8.0 with Tris-HCl and 9.0 with glycine-NaOH buffer. The system requires Mg 2+ and is stimulated by ATP, KCl, sucrose and glycerol; but deoxyribonuclease, polyamines, Na 4 P 2 O 7 , actinomycin D, ethidium bromide and acriflavin inhibit incorporation. 3. Addition of native DNA to the reaction stimulates incorporation 5-fold. DNA synthesis by endogenously and exogenously primed nuclei has been compared. CsCl density gradient centrifugation indicates that endogenously primed reactions synthesize rat liver DNA, but addition of exogenous DNA causes inhibition of endogenous DNA synthesis in favor of the exogenous DNA.

Variability in the nucleic acid binding site size and the amount of single-stranded DNA-binding protein in Escherichia coli

The Escherichia coli single‐stranded DNA binding protein (SSB), essential for DNA replication, recombination and repair, can undergo a thermally induced irreversible conformational change which does not eliminate its biological activity, but changes the number of nucleotides it covers (binding site size) when binding to a single‐stranded nucleic acid lattice. The binding site size of native and conformationally changed SSB was also found to be a function of the molecular mass of the polynucleotide, an observation which is unusual for single‐stranded DNA binding proteins and will greatly affect the affinity relationship of this protein for nucleic acids. A radioimmunoassay used to quantitate in SSB level in cells revealed the number of SSB tetramers to be larger than initial estimates by a factor of as much as six. All these data suggest that the biological role of SSB and its mechanism of action is by far more complex than originally assumed.

Purification and properties of DNA polymerase-β from guinea pig liver

Abstract Deoxyribonucleic acid polymerase-β (EC 2.7.7.7) has been purified over 100 000-fold from a whole cell extract of guinea pig liver. The enzyme yields a single stainable band when subjected to non-denaturing polyacrylamide gel electrophoresis, and this band corresponds to the DNA polymerase activity when a sister gel is sliced and assayed. The final fraction has a specific activity of 21 000 units/mg; this value can be increased significantly by addition of various components, including glycols, polyamines or any of several protein factors which can be purified from the crude extract. The DNA polymerase-β lacks detectable exonuclease or endonuclease activity, has an alkaline pH optimum and has a requirement for all four deoxyribonucleoside triphosphates, a divalent cation and a primer-template for maximal activity. While activated DNA is the preferred primer-template, the enzyme is capable of utilizing native and denatured DNA as well as several synthetic polynucleotides as primer-templates. The latter are especially effective when manganese is the divalent cation. Magnesium, at 10 mM, is the preferred divalent cation when activated DNA is used. Manganese, and to a lesser extent cobalt, can substitute for magnesium, while zinc and calcium cannot. The β-polymerase has a half-life of 10 min at 40°C and this is increased in the presence of either DNA or NaCl. The enzyme is stimulated by glycols, polyamines and NaCl or KCl, and is inhibited by several known inhibitors of DNA polymerase activity including o-phenanthroline, heparin, organic solvents and sulfhydryl blocking agents. Guinea pig liver DNA polymerase-β is remarkably similar to the rat Novikoff hepatoma β-polymerase with respect to its isoelectric point of 8.4 and its molecular weight of 32 000 as determined by sucrose gradient centrifugation under high or low salt conditions or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This similarity is further extended to the removal, at the final step in purification, of a protein capable of stimulating the homogeneous enzyme. Removal of this protein could explain the lower molecular weight of the guinea pig and other rodent-derived β-polymerases, when compared to the β-polymerases from other systems.

Partial purification of a chloroplast DNA polymerase from Euglena gracilis

Abstract A single DNA polymerase has been purified 965 fold from isolated chloroplasts of Euglena gracilis with a yield of 53%. The isolation methods include solubilization of the enzyme with 1M NaCl, ammonium sulfate precipitation, DNA affinity and DEAE-cellulose chromatography. The enzyme requires all four deoxynucleotide triphosphates, magnesium and denatured DNA for maximal activity. The chloroplast DNA polymerase is free of contaminating nucleases and phosphatases, has a sharp pH optimum at pH 7.2 and magnesium optimum of 6mM.

Subcellular localization of high and low molecular weight DNA polymerases of rat liver

Abstract DNA polymerases from isolated rat liver organelles have been characterized by sucrose gradient centrifugation and gel filtration. Mitochondrial DNA polymerase has a molecular weight of about 150,000. The nuclear DNA polymerase has a molecular weight of about 35,000.

The relative rate of synthesis and levels of single-stranded DNA binding protein during induction of SOS repair in Escherichia coli

SummaryInduction of the SOS response in Escherichia coli results in an increase in the relative rate of synthesis of single-stranded DNA binding protein (SSB). In contrast to RecA protein, this increase is slow and does not lead to higher SSB levels. The significance of ssb induction to SOS repair is discussed.

An EPR study to determine the relative nucleic acid binding affinity of single-stranded DNA-binding protein from Escherichia coli

A direct quantitative determination by EPR of the nucleic acid binding affinity relationship of the single-stranded DNA-binding protein (SSB) from Escherichia coli at close to physiological NaCl concentration is reported. Titrations of (DUAP, dT)n, an enzymatically spin-labeled (dT)n, with SSB in 20 mM Tris-HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton with either low (5 mM), intermediate (125 mM) or high 200 mM) NaCl content, reveal the formation of a high nucleic acid density complex with a binding stoichiometry (s) of 60 to 75 nucleotides per SSB tetramer. Reverse titrations, achieved by adding (DUAP, dT)n to SSB-containing solutions, form a low nucleic acid density complex with an s = 25 to 35 in the buffer with low NaCl content (5 mM NaCl). The complex with an s = 25 to 35 is converted to the high nucleic acid density complex by increasing the NaCl content to 200 mM. It is, therefore, metastable and forms only under reverse titration conditions in low NaCl. The relative apparent affinity constant Kapp of SSB for various unlabeled single-stranded nucleic acids was determined by EPR competition experiments with spin-labeled nucleic acids as macromolecular probes in the presence of the high nucleic acid density complex. The Kapp of SSB exhibits the greatest affinity for (dT)n as was previously found for T4 gene 32 protein (Bobst, A.M., Langemeier, P.W., Warwick-Koochaki, P.E., Bobst, E.V. and Ireland, J.C. (1982) J. Biol. Chem. 257, 6184) and gene 5 protein (Bobst, A.M., Ireland, J.C. and Bobst, E.V. (1984) J. Biol. Chem. 259, 2130) by EPR competition assays. In contrast, however, SSB does not display several orders of magnitude greater affinity for (dT)n than for other single stranded DNAs as is the case with both gene 5 and T4 gene 32 protein. The relative Kapp values for SSB in the above buffer with 125 mM NaCl are: Kapp(dT)n = 4KappfdDNA = 40Kapp(dA)n = 200Kapp(A)n.

Interaction of DNA Accessory Proteins with DNA Polymerase β of the Novikoff Hepatoma

Replication and repair require the concerted action of a DNA polymerase with several accessory proteins. In prokaryotes the interaction of such proteins in replication has been well documented (1). With DNA repair, less is known, although with the recent progress of the uvr system of Escherichia coli we can anticipate a better understanding of excision-repair in bacteria (2). In eukaryotes there is a wealth of information on polymerases, but little progress has been made with mechanistic studies of accessory proteins for replication or repair.

CHARACTERIZATION OF MAMMALIAN DNASE V, A DOUBLE-STRAND BIDIRECTIONAL EXONUCLEASE WHICH INTERACTS WITH DNA POLYMERASE-β OF THE NOVIKOFF HEPATOMA

ABSTRACT Novikoff hepatoma factor IV, a protein identified by its ability to stimulate the β-polymerase, has now been purified to homogeneity. This 12,000 dalton polypeptide has both 3′→′ and 5′→3′ exonuclease activity on double-stranded substrates and forms a specific complex with the β-polymerase in a 1:1 stoichiometry. Some of the properties of this enzyme are described. Since this enzyme differs from other mammalian nucleases, we have named it DNase V.