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Biomedical subjects

R R Meyer

Publications and source records attributed to R R Meyer.

At least 19 recordsLinked to original sources
PubMed1994

Mammalian DNA polymerases alpha, beta, gamma, delta, and epsilon incorporate fialuridine (FIAU) monophosphate into DNA and are inhibited competitively by FIAU Triphosphate.

Fialuridine [FIAU, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- iodouridine] was used in clinical trials for chronic hepatitis B virus infection and was extremely toxic. Evidence suggested targets of FIAU toxicity included mitochondria, but toxic mechanisms were unclear. Since FIAU is a thymidine analog, we reasoned that triphosphorylated FIAU (FIAUTP) could be incorporated into mitochondrial DNA by DNA pol-gamma and into genomic DNA by DNA polymerases alpha, beta, delta, and epsilon. All five purified mammalian DNA polymerases incorporated FIAUMP into the nascent DNA chain during in vitro DNA synthesis. When FIAUTP was substituted for dTTP, oligonucleotide products were generated efficiently by DNA pol-gamma and were similar to those generated in the presence of the four normal dNTPs. In contrast, oligonucleotide products generated by the four nuclear DNA polymerases in the presence of FIAUTP were significantly reduced in length relative to those generated in the presence of dTTP. In parallel kinetic assays, FIAUTP competitively inhibited the accumulation of radiolabeled dTTP into DNA by DNA pol-gamma. The Ki with DNA pol-gamma was 0.04 microM, the lowest Ki among the mammalian DNA polymerases. Competition between FIAUTP and dTTP and the relative ease of accumulation of FIAUMP in mitochondrial DNA by DNA pol-gamma in vitro together may relate to clinical FIAU toxicity.

Animals↗
PubMed1994

Cardiac mitochondrial DNA polymerase-gamma is inhibited competitively and noncompetitively by phosphorylated zidovudine.

Zidovudine (azidothymidine [AZT]) inhibits human immunodeficiency virus replication and reduces the severity of acquired immunodeficiency syndrome. A limiting side effect of AZT is a mitochondrial cardiac and skeletal myopathy in which the pharmacologically active derivative of AZT (AZT triphosphate) plays a critical role. The present study determined biochemical mechanisms of AZT-induced mitochondrial toxicity and identified AZT triphosphate as an inhibitor of DNA polymerase-gamma in vitro. Inhibition kinetics were defined using purified bovine cardiac mitochondrial DNA polymerase-gamma and AZT triphosphate in vitro. The Km for deoxythymidine triphosphate was 0.8 +/- 0.3 mumol/L. AZT triphosphate incubation with DNA polymerase-gamma in vitro resulted in mixed kinetics with a competitive Ki of 1.8 +/- 0.2 mumol/L and a noncompetitive Ki' of 6.8 +/- 1.7 mumol/L. These Ki and Ki' values were strikingly higher than values for retroviral reverse transcriptase but lower than values for other cellular DNA polymerases. These data support previous molecular and morphological findings in clinical AZT mitochondrial myopathy and in models of AZT myopathy in vivo. Biochemical findings suggest that inhibition of mitochondrial DNA polymerase-gamma may be integral to the pathogenesis of AZT-induced myopathy.

Animals↗
PubMed1992

Structure-function analysis of DNA polymerase-beta using monoclonal antibodies: identification of a putative nucleotide binding domain.

DNA polymerase-beta was purified from Novikoff hepatoma and used as an antigen in an in vitro immunization system to produce monoclonal antibodies. These reagents surprisingly showed cross-reactivity to a number of proteins, including several DNA polymerases. Nearly all of these proteins possess nucleotide binding sites, which suggested the potential value of using the monoclonals to elucidate structure-function relationships within polymerase-beta. Furthermore, these antibodies were able to partially neutralize (40-50%) polymerase-beta activity, and this effect could be blocked by dNTP1 but not by dNMP or rNTP. The limited neutralization phenomenon is at least partially explained by the weak binding affinity of these antibodies. Scatchard analysis of immunoprecipitation data predicted a Kd of 1.8 x 10(-8) M. Epitope mapping studies showed that the region of polymerase-beta recognized by one of the monoclonal antibodies is within residues 235-335, and sequence homology studies indicated that the epitope is probably located in the region of amino acids 283-320. At least a portion of this area, namely residues 301-308 and 311-315, appears to be part of a nucleotide binding domain which has sequence homology with a portion of the highly conserved ATP binding site in adenylate kinase.

Amino Acid Sequence↗
PubMed1992

Interaction of the heat shock protein GroEL of Escherichia coli with single-stranded DNA-binding protein: suppression of ssb-113 by groEL46.

Previous studies from our laboratory have shown that an allele of the heat shock protein GroEL (groEL411) is able to specifically suppress some of the physiological defects of the single-stranded DNA-binding protein mutation ssb-1. A search for additional alleles of the groE genes which may act as suppressors for ssb mutations has led to the identification of groEL46 as a specific suppressor of ssb-113. It has very little or no effect on ssb-1 or ssb-3. All of the physiological defects of ssb-113, including temperature-sensitive growth, temperature-sensitive DNA synthesis, sensitivity to UV irradiation, methyl methanesulfonate, and bleomycin, and reduced recombinational capacity, are restored to wild-type levels. The ssb-113 allele, however, is unable to restore sensitivity of groEL46 cells to phage lambda. The mechanism of suppression of ssb-113 by groEL46 appears to differ from that of ssb-1 by groEL411. The data suggest that GroEL may interact with single-stranded DNA-binding protein in more than one domain.

Bacterial Proteins↗
PubMed1991

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.

DNA, Bacterial↗
PubMed1990

The single-stranded DNA-binding protein of Escherichia coli.

The single-stranded DNA-binding protein (SSB) of Escherichia coli is involved in all aspects of DNA metabolism: replication, repair, and recombination. In solution, the protein exists as a homotetramer of 18,843-kilodalton subunits. As it binds tightly and cooperatively to single-stranded DNA, it has become a prototypic model protein for studying protein-nucleic acid interactions. The sequences of the gene and protein are known, and the functional domains of subunit interaction, DNA binding, and protein-protein interactions have been probed by structure-function analyses of various mutations. The ssb gene has three promoters, one of which is inducible because it lies only two nucleotides from the LexA-binding site of the adjacent uvrA gene. Induction of the SOS response, however, does not lead to significant increases in SSB levels. The binding protein has several functions in DNA replication, including enhancement of helix destabilization by DNA helicases, prevention of reannealing of the single strands and protection from nuclease digestion, organization and stabilization of replication origins, primosome assembly, priming specificity, enhancement of replication fidelity, enhancement of polymerase processivity, and promotion of polymerase binding to the template. E. coli SSB is required for methyl-directed mismatch repair, induction of the SOS response, and recombinational repair. During recombination, SSB interacts with the RecBCD enzyme to find Chi sites, promotes binding of RecA protein, and promotes strand uptake.

Amino Acid Sequence↗
PubMed1988

Interaction of a folded chromosome-associated protein with single-stranded DNA-binding protein of Escherichia coli, identified by affinity chromatography.

A single-stranded DNA-binding protein (SSB) affinity column was prepared by optimizing the coupling of Escherichia coli single-stranded DNA-binding protein to Affi-Gel 10. The bound SSB retained its ability to specifically bind single-stranded DNA. When nuclease-treated cell extracts were incubated with the SSB beads overnight at 4 degrees C, a major protein of Mr = 25,000 was bound. At shorter incubation times, two additional proteins of Mr = 32,000 and 36,000 were also detected. In the absence of nuclease treatment, eight additional proteins ranging from Mr = 14,000 to 160,000 also bound to the affinity column. The major Mr = 25,000 protein has been shown to be a folded chromosome-associated protein. Its binding to SSB is strongly enhanced by the addition of DNA polymerase III or DNA polymerase III holoenzyme.

Bacterial Proteins↗
PubMed1988

Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma.

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.

Adenine Nucleotides↗
PubMed1988

Suppression of the Escherichia coli ssb-1 mutation by an allele of groEL.

A series of spontaneous suppressors to the temperature-sensitive phenotype of the single-stranded DNA-binding protein mutation ssb-1 were isolated. A genomic library of EcoRI fragments from one of these suppressor strains was prepared by using pBR325 as the cloning vector. A 10.0-kilobase class of inserts was identified as carrying the ssb-1 gene itself. A second class of 8.3-kilobase inserts was shown to contain the groE region by (i) restriction analysis, (ii) Southern hybridization of the 8.3-kilobase insert to groE+ DNA, and (iii) identification of the gene products by similar migration on polyacrylamide gels. Subcloning demonstrated that an intact mutant groEL gene was necessary for suppression and that plasmids carrying the 8.3-kilobase insert could suppress mutants carrying groES- but not groEL- genes for phage lambda growth. The suppressor, designated as groEL411, was specific for the ssb-1 allele. In ssb-1 groEL411 cells, DNA synthesis stopped after a shift to 42.5 degrees C but rapidly recovered within minutes. The data suggest a direct interaction between the single-stranded DNA-binding protein and GroEL proteins in DNA replication.

Bacterial Proteins↗
PubMed1986

Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus.

An inorganic pyrophosphatase was purified over 600-fold to homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme is a tetramer of Mr = 84,000, has a sedimentation coefficient of 5.8S, a Stokes radius of 3.5 nm, and an isoelectric point of 5.7. Like the enzyme of Escherichia coli, the pyrophosphatase appears to be made constitutively. The pH and temperature optima are 8.3 and 80 degrees C, respectively. The Km for PPi is 0.6 mM. A divalent cation is essential, with Mg2+ preferred. The enzyme uses only PPi as a substrate.

Centrifugation, Density Gradient↗
PubMed1985

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.

Binding Sites↗
PubMed1984

A new DNA-dependent ATPase from Escherichia coli. Purification and characterization of ATPase IV.

A new DNA-dependent ATPase named ATPase IV has been purified to apparent homogeneity from Escherichia coli as a by-product of DNA polymerase III purification. The enzyme has a specific activity of 360 mumol of ATP hydrolyzed per min/mg of protein. The purified enzyme exists as monomer with a molecular weight of 81,000. It sediments in a glycerol gradient as a single species of 4.5 S. The enzyme has considerable activity at 0 degree C and has a Q10 of 3.8. In the presence of a DNA effector and magnesium ion, the enzyme will hydrolyze ATP, dATP, GTP, or dGTP to a nucleoside diphosphate plus orthophosphate with a Km of 0.20, 0.50, 0.60, and 1.30 mM, respectively. The guanine nucleotides, however, are only 25-35% as effective as substrates compared with the adenine nucleotides. ATPase IV shows strong substrate inhibition by ATP, but not dATP, above 0.2 mM. The polynucleotide effector requirement can be satisfied by either single-stranded or double-stranded DNA. The enzyme binds the effector very tightly with a Km of 3 X 10(-8) M (nucleotide) for G4 DNA. The enzyme is inhibited by E. coli single-stranded DNA-binding protein, a variety of ATP analogues and N-ethylmaleimide. The relationship of ATPase IV to DNA polymerase III holoenzyme is discussed.

Adenosine Triphosphatases↗
PubMed1983

A novel single-stranded DNA-binding protein from the Novikoff hepatoma which stimulates DNA polymerase beta. Purification and general characterization.

We have isolated and purified to homogeneity a novel single-stranded DNA-binding protein from the Novikoff hepatoma. This protein is distinguished from other eukaryotic DNA-binding proteins by binding weakly, but cooperatively, to single-stranded DNA, by its ability to partially destabilize a double helix at 37 degrees C, and by its ability to stimulate DNA polymerase beta. The protein exists as a globular monomer of Mr = 48,000 and is capable of binding 45-49 nucleotides. It does not form a complex with the polymerase, but binds the DNA template, allowing an increased rate and extent of DNA synthesis. The enhancement of synthesis is greatest with larger gap-sized templates and with low polymerase concentrations. The mechanism of stimulation is thought to be due largely to placing the template strand into a conformation that facilitates rapid polymerization rather than strand displacement in advance of the polymerase. This protein has been named SSB-48.

Amino Acids↗
PubMed1982

Deoxyribonucleic acid dependent adenosinetriphosphatases from the Novikoff hepatoma. Characterization of a homogeneous adenosinetriphosphatase that stimulates DNA polymerase beta.

Five chromatographically distinct DNA-dependent ATPase activities have been identified in high salt-detergent extracts of the Novikoff hepatoma. One of these, ATPase III, has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis and has a specific activity of 12 mumol of ATP hydrolyzed min-1 (mg of protein)-1. The enzyme, a dimer of Mr 65000 subunits, has a sedimentation coefficient of 7.0 S in both high salt and low salt, a Stokes radius of 43 A, and a frictional coefficient of 1.31. In the presence of Mg2+ ion and a polynucleotide effector, the enzyme catalyzes hydrolysis of ATP or dATP to a diphosphate with a Km of 206 microM and 110 microM, respectively, for the two substrates. Although single-stranded effectors are preferred, the enzyme has significant activity with double-stranded effectors. The Km for effector is 0.4 microM (nucleotide). The analogues adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), dideoxyadenosine triphosphate (ddATP), and adenosine 5'-(alpha, beta-methylenetriphosphate) (alpha, beta-Me-ATP) are competitive inhibitors of the enzyme while adenosine tetraphosphate (ATP-P), 8-bromoadenosine 5'-triphosphate (8-Br-ATP), 5'-adenylyl imidodiphosphate (AMP-PNP), and adenosine 5'-(beta, gamma-methylenetriphosphate) (beta, gamma-Me-ATP) do not inhibit. The enzyme is insensitive to nalidixic acid, novobiocin, and berenil but is sensitive to N-ethylmaleimide. ATPase III is capable of stimulating DNA polymerase beta on duplex DNA, but this effect is abolished in the presence of ATP gamma S. Polymerase stimulation is further enhanced in the presence of a single-stranded DNA-binding protein. These data suggest that ATPase III may play a role in DNA repair.

Adenosine Triphosphatases↗
PubMed1982

Influence of single-stranded DNA-binding protein on recA induction in Escherichia coli.

Two ssb mutants of Escherichia coli, which carry a lesion in the single-strand DNA-binding protein (SSB), are sensitive to UV-irradiation. We have investigated the influence of SSB on the "SOS" repair pathway by examining the levels of recA protein synthesis. These strains fail to induce normal levels of recA protein after treatment with nalidixic acid or ultraviolet light. The level of recA protein synthesis in wild-type cells is about three times greater than ssb cells. This deficiency in ssb mutants occurs in all strains and at all temperatures tested (30-41.5 degrees). In contrast, the ssb-1 mutation has no effect on temperature-induced recA induction in a recA441 (tif-1) strain. Cells carrying ssb+ plasmids and overproducing normal DNA-binding protein surprisingly are moderately UV-sensitive and have reduced levels of recA protein synthesis. Together these results establish that single-strand DNA-binding protein is involved in the induction of recA, and accounts, at least in part, for the UV sensitivity of ssb mutants. Three possible mechanisms to explain the role of SSB are discussed.

Bacterial Proteins↗
PubMed1982

The product of the lexC gene of Escherichia coli is single-stranded DNA-binding protein.

Extracts from lexC113 cells could not support phage G4 DNA-dependent replication unless supplemented with single-stranded DNA-binding protein. Purified lexC113 binding protein supported synthesis in a reconstituted replication assay, using purified proteins at 30 but not at 42 degrees C, indicating that the product of the lexC113 gene is an altered single-stranded DNA-binding protein.

Bacterial Proteins↗