Biological consequences of a reduction in the non-target DNA scanning capacity of a DNA repair enzyme

Numerous DNA-interactive proteins have been shown to locate specific sequences within large domains of non-target DNA in vitro and in vivo by a one-dimensional diffusion mechanism; however, the biological significance of this process has not been evaluated. We have examined the biological consequences of sliding for the pyrimidine dimer-specific DNA repair enzyme T4 endonuclease V, an enzyme which scans non-target DNA both in vitro and in vivo. An endonuclease V mutant was constructed whose only altered biochemical characteristic, measured in vitro, was a loss in its ability to slide on non-target DNA. In contrast to the native enzyme, when the mutated endonuclease V was expressed in DNA repair-deficient Escherichia coli, no enhanced ultraviolet survival was conferred. These results suggest that the mechanisms which DNA-interactive proteins employ to enhance the probability of locating their target sequences are of significant biological importance.     

Inhibition of bacterial aminopropyltransferases by S-adenosyl-1,8-diamino-3-thiooctane and by dicyclohexylamine

Bacterial aminopropyltransferases from Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa were strongly inhibited by S-adenosyl-1,8-diamino-3-thiooctane (AdoDATO) and by dicyclohexylamine. The sensitivity to these drugs in vitro was comparable to that of mammalian spermidine synthase, but AdoDATO was much less potent in reducing spermidine content in the bacteria than in mammalian cells. Although AdoDATO was a stronger inhibitor than dicyclohexylamine in vitro, dicyclohexylamine was more active in reducing bacterial spermidine levels in vivo, suggesting that it is taken up better or is more stable in the cell and is the preferable compound for in vivo studies in microorganisms. The strong inhibition of spermidine synthases by AdoDATO which is a transition state analog supports the concept that these enzymes proceed by a single displacement reaction, rather than by a ping-pong mechanism.     

Effects of polyamines and methylglyoxal bis(guanylhydrazone) on Escherichia coli ribonucleic acid polymerase and the template activity of hepatic cell nuclei in vitro

Escherichia coli RNA polymerase was assayed with 4 mM Mg2+ and 1 mM Mn2+ using native DNA, heat-denatured DNA, histone-nucleate and isolated rat liver nuclei as the template source. With purified DNA and either or both divalent metal ions, 0.1--5 mM amine stimulated enzyme activity. Spermidine resulted in the greatest stimulation (1.7-fold at 5 mM); whereas, spermine or methylglyoxal bis(guanylhydrazone) first stimulated, then above 3 mM inhibited, the reaction. The addition of unfractionated histone to purified DNA inhibited the reaction by 90%. The subsequent addition of amines resulted in a slight stimulation in incorporation (1.5-fold) in the range of 1--3 mM amine. Alternatively, when enzyme was combined with DNA before histone, only a 20% inhibition was observed and this could be completely prevented by 3 mM spermidine. The addition of amines to isolated nuclei resulted in marked alterations in ultrastructure and Mg2+ content; however, relatively small effects on RNA polymerase activity were observed. With the E. coli enzyme, 0.1--1.0 mM amine stimulated RNA synthesis (1.5-fold) whereas, none of the amines stimulated endogeneous activity in the absence or presence of 300 mM (NH4)2SO4.     

Effect of various polyamine analogs on in vitro polypeptide synthesis

Various polyamine analogs were examined for their ability to stimulate and to function as sparing agents for the Mg2+ requirement in polypeptide synthesis at various temperatures in Escherichia coli (37 and 47 degrees C) and the extremely thermophilic Thermus thermophilus (60 and 70 degrees C) cell-free systems. The optimal concentration of each polyamine analog increased as the incubation temperature was elevated. At a fixed temperature, the optimal concentration of polyamine analogs was in the order diamines greater than triamines greater than tetraamines greater than pentaamines. All diamines tested stimulated polypeptide synthesis almost equally but lowered the optimal Mg2+ concentration in the order diaminopropane greater than putrescine greater than cadaverine. The degree of diamine stimulation was maximal at 37 degrees C. The effects of three triamines were very similar in the E. coli system but in the T. thermophilus system spermidine was most effective in stimulation of polypeptide synthesis. From the results of experiments using tetraamines and pentaamines, it was deduced that the presence of both aminobutyl and aminopropyl groups in polyamine analogs is important for stimulation of polypeptide synthesis. In the E. coli system, triamines were the most effective polyamines for stimulation of polyphenylalanine synthesis at both 37 and 47 degrees C, while, in the T. thermophilus system, thermospermine, a tetraamine, was most effective at 60 degrees C and 3,4,4,3-pentaamine was most effective at 70 degrees C.     

The DNA unwinding reaction catalyzed by Rep protein is facilitated by an RHSP-DNA interaction.

The unwinding reaction catalyzed by the Escherichia coli Rep protein is stimulated by a small 15 kDa protein called Rep helicase stimulatory protein (RHSP)(1). The RHSP-stimulated unwinding reaction catalyzed by Rep protein proceeded at a rapid rate after a time lag of 1-2 min at 37 degrees C. This time lag was eliminated by preincubating RHSP with the DNA substrate, indicating that stimulation resulted from an interaction between RHSP and DNA. RHSP was shown to increase the rate as well as the extent of the unwinding reaction catalyzed by Rep protein. RHSP bound both single- and double-stranded DNA with apparent equal affinity, forming an unusually stable complex. Electron microscopy illustrated that the RHSP-DNA complex consisted of large protein aggregates bound to DNA forming a highly condensed, aggregated DNA-protein complex. The protein aggregates were not observed in the absence of DNA and appeared to form cooperatively in the presence of DNA. NH2-terminal amino acid sequence analysis suggested that RHSP was identical to E. coli ribosomal-protein L14. Binding assays showed that the interaction between RHSP and rRNA was similar to the RHSP-DNA interaction. Several models are put forth to explain the stimulation of the unwinding reaction catalyzed by Rep protein. In addition, the potential physiological significance of the RHSP-stimulated Rep protein unwinding reaction is discussed.     

On the roles of magnesium and spermidine in the isoleucyl-tRNA synthetase reaction. Analysis of the reaction mechanism by total rate equations

The reaction of isoleucyl-tRNA synthetase from Escherichia coli B was analysed by deriving total steady-state rate equations for the ATP/PPi exchange reaction and for the aminoacylation of tRNA, and by fitting these rate equations to series of experimental results. The analysis suggests that (a) a Mg2+ inhibits the aminoacylation of tRNA but not the activation of the amino acid. In the chosen mechanism, this enzyme-bound Mg2+ is required at the activation step. (b) Another Mg2+ is required at ATP, but the MgATP apparently can be replaced by the spermidine.ATP complex. Spermidine.ATP is a weaker substrate. The role of spermidine.ATP is especially suggested by the relative rates of the aminoacylation of tRNA when the spermidine and magnesium concentrations are varied. The aminoacylation measurements still suggest that (c) two (or more) Mg2+ are bound to the tRNA molecule and are required for enzyme activity at the transfer step, and that these Mg2+ can be replaced by spermidines.     

Methylglyoxal bis(guanylhydrazone) elimination of polyamine effects on protein synthesis

The effect of methylglyoxal bis(guanylhydrazone) (MGBG), a structural analog of polyamines, on protein synthesis has been studied in the presence and absence of spermidine. The spermidine stimulation of polyphenylalanine- and MS2 RNA-directed RNA replicase synthesis in an Escherichia coli cell-free system and of globin synthesis in a rabbit reticulocyte cell-free system disappeared with the addition of MGBG. The spermidine reduction of misincorporation of leucine during polyphenylalanine synthesis in both E. coli and wheat germ cell-free systems was also disturbed by MGBG. MGBG noncompetitively interfered with polyamine stimulation of polyphenylalanine and globin synthesis, suggesting that MGBG could bind to both RNA and the complex of RNA and polyamine. MGBG was preferentially bound to ribosomal RNA among ribosomal RNA, poly(U), and calf thymus DNA, and strongly inhibited the amount of polyamine bound to ribosomal RNA. These results suggest that MGBG elimination of polyamine effects on protein synthesis may occur through the disturbance of polyamine binding to ribosomal RNA.     

Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim.

At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes.     

Stimulation of deoxyribonucleic acid replication fork movement by spermidine analogs in polyamine-deficient Escherichia coli.

We examined the rate of deoxyribonucleic acid (DNA) replication fork movement in polyamine-deficient cells of Escherichia coli by two independent techniques. DNA autoradiography was used to directly visualize the length of DNA produced during a given time interval, and replication rates were calculated. The amount of DNA synthesized after blocking protein synthesis also allowed calculation of replication rates. We found that the DNA chain elongation rate in polyamine-deficient cells was about half that of putrescine- or spermidine-supplemented cells. We also found that spermidine homologs of increasing chain length, when present at equal intracellular concentrations, exhibited a decreasing ability to support growth and the rate of DNA replication fork movement. The kinetics of recovery of DNA synthesis from the polyamine-deficient state were also investigated. A new rate of DNA synthesis was reached about 20 min after addition of spermidine to polyamine-limited cells. The rise in the rate of DNA synthesis was preceded by a rise in the intracellular concentration of spermidine.     

Purification and characterization of the integrase from the Haemophilus influenzae bacteriophage HP1; identification of a four-stranded intermediate and the order of strand exchange

The integrase encoded by the temperate phage HP1 promotes the site-specific recombination between DNA sites on its genome (the attP site) and on the genome of the host Haemophilus influenzae (the attB site). The protein has been overproduced in Escherichia coli , and purified to apparent homogeneity. HP1 integrase promotes recombination of supercoiled attP -containing molecules with linear segments with attB sites. Reaction was enhanced by spermidine and by the bacterial DNA-bending protein integration host factor. The rate of recombination showed complex and related dependence upon the integrase concentration and the concentration of the supercoiled attP substrate. These relationships probably originate from the need to assemble a multi-protein complex on the attP DNA. The reaction promoted by HP1 integrase produced a four-stranded initial reaction product in which one pair of DNA strands had undergone transfer while the other pair remained intact. This four-stranded component was produced more rapidly than any product, and its steady-state level was proportional to the overall rate of reaction. This component had the kinetic and structural properties of an intermediate in the recombination reaction. The existence of this intermediate was used to determine that the two strand exchanges required for recombination of the duplex substrates proceed in a defined order.     

Structural specificity of the spermidine requirement of an Escherichia coli auxotroph.

A homologous series of spermidine analogs was synthesized with the general structure NH3+ (CH2)nNH2+(CH2)3NH3+, where spermidine has n = 4. The influence of these compounds on growth and on the syntheses of protein and messenger ribonucleic acid was examined in a spermidine auxotroph of Escherichia coli. All of the homologs tested were taken up by the cells to an intracellular level equivalent to the level of spermidine which gives optimal growth. With increasing chain length of the homologs, there was reduced ability to stimulate growth. The homologs with n = 7 and n = 8 were essentially inactive. A similar specificity was observed when the ability of the homologs to restore the rates of protein and messenger ribonucleic acid chain elongation was compared to that of spermidine. These results suggest that a definite spatial arrangement of the amino groups of spermidine is required for productive interaction at its intracellular site(s) of action.     

A high-throughput assay for the adenylation reaction of bacterial DNA ligase

DNA ligase catalyzes the closure of single-strand nicks in double-stranded DNA that arise during replication and recombination. Inhibition of bacterial ligase is expected to cause chromosome degradation and cell death, making it an attractive target for new antibacterials. The prototypical bacterial ligase couples the hydrolysis of NAD(+) to phosphodiester bond formation between an adjacent 3'OH and 5'-terminal phosphate of nicked duplex DNA. The first step is the reversible formation of a ligase-adenylate from the reaction between apoenzyme and NAD(+). Inhibitors that compete with NAD(+) are expected to be bacterial specific because eukaryotic DNA ligases use ATP and differ in the sequence composition of their adenylation domain. We report here a high-throughput assay that measures the adenylation reaction specifically by monitoring ligase-AMP formation via scintillation proximity technologies. Escherichia coli DNA ligase was biotinylated in vivo; after reaction with radiolabeled NAD(+), ligase-[(3)H]AMP could be captured onto the streptavidin-coated surface of the solid scintillant. The method was ideal for high-throughput screening because it required minimal manipulations and generated a robust signal with minimal scatter. Certain adenosine analogs were found to inhibit the adenylation assay and had similar potency of inhibition in a DNA ligation assay.     

Purification of Escherichia coli DNA photolyase

Escherichia coli photolyase is a DNA repair enzyme which monomerizes pyrimidine dimers, the major UV photoproducts in DNA, to pyrimidines in a light-dependent reaction. We recently described the construction of a tac-phr plasmid that greatly overproduces the enzyme (Sancar, G. B., Smith, F. W., and Sancar, A. (1983) Nucleic Acids Res. 11, 6667-6678). Using a strain carrying the overproducing plasmid as the starting material, we have developed a purification procedure that yields several milligrams of apparently homogeneous enzyme. The purified protein is a single polypeptide that has an apparent Mr of 49,000 under both denaturing and nondenaturing conditions. The enzyme has no requirement for divalent cations and it restores the biological activity of irradiated DNA only in the presence of photoreactivating light. The purified photolyase has a turnover number of 2.4 dimers/molecule/min; this value agrees well with the in vivo rate of photoreactivation in E. coli.     

Differential stimulation by polyamines of phage RNA-directed synthesis of proteins

The effect of polyamines on Q beta and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied. With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA replicase greater than A protein, while the synthesis of coat protein was not stimulated significantly by spermidine. The synthesis of RNA replicase was stimulated by 1 mM spermidine approx. 8-fold. From the results of Q beta RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.     

Genetic control of the protective effect of spermidine in Escherichia coli cells as affected by mitomycin C

The lethal action of mitomycin C and the effect of mutual treatment with mitomycin C and spermidine on Escherichia coli were studied. DNA repair in cells treated with mitomycin C was shown to have some differences, as compared to that of UV-induced pyrimidine dimers. The presence of the additive sbcB mutation increases the resistance of wild-type bacteria as well as of recBrecC and recF mutants to the lethal action of mytomicin C. Preliminary treatment of bacteria with spermidine increases resistance to the lethal action of the mutagen in wild-type bacteria as well as uvrB, recBrecC and sbcB strains. However, no such effect was observed in recF, recFsbcB and uvrE strains. The data suggest that the protective action of spermidine may be connected with stimulation of RecF-pathway of postreplication repair.     

DNA-dependent in vitro synthesis of enzymes of the galactose operon of Escherichia coli

Summary Two active enzymes of the galactose operon of Escherichia coli, uridyl transferase and galactokinase have been synthesized with high yields in a DNA dependent system for protein synthesis. The unspecific blank values amount to less than two percent of the rate obtained under optimal conditions and permit the accurate determination of even a small fraction of the maximum synthesis rate. Therefore this system provides a sensitive assay for the biological activity of DNA that contains the intact galactose operon of Escherichia coli.The synthesis of these galactose enzymes is to a high extent dependent on the presence of cyclic adenosine-3:5-monophosphate.D-fucose, known as an inducer of the galactose operon in vivo, stimulates the synthesis of galactokinase, indicating that the repressor of the galactose operon in active under these conditions. This stimulation is not observed, if the bacterial extract is prepared from a strain defective for the galactose repressor or if the DNA carries an operator constitutive mutation in the galactose operon. Therefore the stimulation by D-fucose is true derepression.     

Structural polymorphism of the RecA protein from the thermophilic bacterium Thermus aquaticus.

The Escherichia coli RecA protein has served as a model for understanding protein-catalyzed homologous recombination, both in vitro and in vivo. Although RecA proteins have now been sequenced from over 60 different bacteria, almost all of our structural knowledge about RecA has come from studies of the E. coli protein. We have used electron microscopy and image analysis to examine three different structures formed by the RecA protein from the thermophilic bacterium Thermus aquaticus. This protein has previously been shown to catalyze an in vitro strand exchange reaction at an optimal temperature of about 60 degrees C. We show that the active filament formed by the T. aquaticus RecA on DNA in the presence of a nucleotide cofactor is extremely similar to the filament formed by the E. coli protein, including the extension of DNA to a 5.1-A rise per base pair within this filament. This parameter appears highly conserved through evolution, as it has been observed for the eukaryotic RecA analogs as well. We have also characterized bundles of filaments formed by the T. aquaticus RecA in the absence of both DNA and nucleotide cofactor, as well as hexameric rings of the protein formed under all conditions examined. The bundles display a very large plasticity of mass within the RecA filament, as well as showing a polymorphism in filament-filament contacts that may be important to understanding mutations that affect surface residues on the RecA filament.