Spermine Condenses DNA,but Not RNA Duplexes

Interactions between the polyamine spermine and nucleic acids drive important cellular processes. Spermine condenses DNA and some RNAs, such as poly(rA):poly(rU). A large fraction of the spermine present in cells is bound to RNA but apparently does not condense it. Here, we study the effect of spermine binding to short duplex RNA and DNA, and compare our findings with predictions of molecular-dynamics simulations. When small numbers of spermine are introduced, RNA with a designed sequence containing a mixture of 14 GC pairs and 11 AU pairs resists condensation relative to DNA of an equivalent sequence or to 25 bp poly(rA):poly(rU) RNA. A comparison of wide-angle x-ray scattering profiles with simulation results suggests that spermine is sequestered deep within the major groove of mixed-sequence RNA. This prevents condensation by limiting opportunities to bridge to other molecules and stabilizes the RNA by locking it into a particular conformation. In contrast, for DNA, simulations suggest that spermine binds externally to the duplex, offering opportunities for intermolecular interaction. The goal of this study is to explain how RNA can remain soluble and available for interaction with other molecules in the cell despite the presence of spermine at concentrations high enough to precipitate DNA.     

Activity of foot-and-mouth disease virus RNA-dependent RNA polymerase in vitro: inhibition by polyamines and poly(amino acid)s

Replication of foot-and-mouth disease virus RNA in vitro is inhibited by high concentrations of the following polyamines in decreasing order of effectiveness: putrescine, spermine, and cadaverine. The basic poly(amino acids), polylysine, polyornithine, and polyarginine, as well as the basic protein salmine, are several orders of magnitude more inhibitory than polyamines. The interaction between polyornithine and foot-and-mouth disease virus RNA and its inhibition of replicase activity are related to the ability of basic polypeptides to bind to the RNA template. The neutral polymer, polysarcosine, and the polyanions, polyglutamic acid and heparin sulfate, do not inhibit replication; however, both polyanions relieve the inhibition by polyamines and polyamino acids. The mode of inhibition by polyamines and poly(amino acids) and the antagonism by heparin is discussed.     

Polyamines and Morphogenesis - Effects of Methylglyoxal-bis(guanylhydrazone)

The effects of methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis were studied on tuberization and cellular polyamine content using in vitro Solanum tuberosum (cv Binjte) plants. When MGBG was added to the culture medium, it produced a partial inhibition of the growth of stems and leaves; it totally blocked rhizogenesis and strongly stimulated tuber formation. Morphogenetic effects of MGBG were correlated to a 40 % decrease in free putrescine, spermidine, spermine content of the leaves and to a 28 % decrease in spermidine titer of the stems. In the tubers, this inhibitor did not change the free polyamine titer but increased by up to 85 % the titer of conjugated putrescine, spermidine, spermine. When the plants were grown in the dark, MGBG produced, like benzyladenine, a stimulation of the rate of tuberization and enhanced the content of conjugated polyamines in the tuber. These results support the hypothesis that polyamines play an important role in the morphogenesis of potato plants. The role of polyamine conjugation in tuber development is discussed.     

Polyamines and nucleic acids during development of the chick embryo

1. A higher concentration of polyamines (spermine, spermidine, putrescine and cadaverine) during development of the chick embryo was observed between the fifth and tenth day of incubation; the concentrations of nucleic acids showed a parallel increase. 2. When spermine (5mumoles) was injected into the yolk sac of embryos at the tenth day of incubation, a high amine-oxidase activity was noted and the spermine and spermidine concentrations were decreased; also, there was a remarkable decrease in RNA and DNA concentrations and a parallel increase in that of total free nucleotides. 3. On the other hand, when iproniazid (16mumoles) was injected there was an inhibition of amine-oxidase activity and a similar increase in the concentrations of spermine and spermidine and of nucleic acids, whereas that of total free nucleotides decreased. 4. Another group of embryos injected with spermine and iproniazid together showed a remarkable increase in spermine and spermidine concentrations and a parallel increase in those of RNA and DNA, and a decrease in that of total free nucleotides.     

Effects of α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on testosterone-induced regeneration of prostate and seminal vesicle in castrated rats

1. Castration of adult rats markedly decreases the amounts of polyamines (putrescine, spermidine and spermine) and of RNA and DNA in the ventral prostate and the seminal vesicle. 2. Daily injections of testosterone propionate to rats castrated 7 days previously increase polyamine and nucleic acid contents more rapidly in the seminal vesicle than in the ventral prostate. 3. After 7 days of androgen treatment, polyamine and nucleic acid contents of the seminal vesicle are significantly higher than those of intact animals. Nucleic acid, but not polyamine, contents return to normal values during the next 4 days of continued treatment. In the prostate, androgen treatment increases polyamine and nucleic acid contents to, but not above, normal values. 4. Repeated doses of alpha-difluoromethylornithine, a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase, totally blocked the testosterone-induced increase of putrescine and spermidine in the ventral prostate and of putrescine in the seminal vesicle. They slowed significantly the accumulation of spermine in the ventral prostate and of spermidine in the seminal vesicle. alpha-Difluoromethylornithine also retarded the testosterone-induced accumulation of RNA in the ventral prostate. However, no clear correlation was apparent between accumulation of polyamines and of nucleic acids in the two organs. 5. alpha-Difluoromethylornithine markedly slows the testosterone-induced weight gain of the prostate, but not of the seminal vesicle. Cytological studies suggest that this effect on the prostate is due to inhibition of the androgen-induced restoration of the secretion content of prostatic acini.     

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.     

Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA

The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide–nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.     

Leishmania spp.: effect of inhibitors on growth and on polyamine and macromolecular syntheses

Ethidium bromide, pentamidine isethionate, and MGBG [methylglyoxal-bis (guanylhydrazone)] inhibited the uptake of radioactive putrescine by leishmanial (Leishmania spp.; Leishmania tropica major; Leishmania mexicana; Leishmania donovani) promastigotes and interfered with their polyamine synthesis. Inhibition was apparent as early as 1 hr after adding these drugs to the parasites at growth-inhibiting concentrations. Ethidium bromide also inhibited the incorporation of radioactive uracil into leishmanial RNA at growth-inhibiting concentrations, while DNA synthesis was inhibited by ethidium bromide at high concentrations after a lag period. MGBG inhibited the synthesis of leishmanial DNA and RNA at growth-inhibiting concentrations.     

Acetylation of spermidine and spermine by rat liver and kidney chromatin

The in vitro enzymatic acetylation of the polyamines, spermidine and spermine, is described. The reaction is catalyzed by chromatin preparations from rat liver and kidney and is dependent on acetyl-CoA. Spermidine, spermine, and putrescine are each converted to the corresponding monoacetyl derivatives. s_0.5 values of 0.5 ± 0.1, 1.0 ± 0.1, and 2.6 ± 0.7 mm (mean ± standard deviation) were obtained for spermidine, spermine, and putrescine, respectively. These values for s_0.5 are similar to the concentrations of polyamines reported for tissues, and therefore, suggest the occurrence of polyamine acetylation in vivo. Evidence is also presented for the metabolism of acetylated polyamines by the 100,000g supernatant fraction of rat liver. The physiological function of polyamine acetylation is unknown, but the possibility of an effect on the association of polyamines with nucleic acids is discussed.     

Action of methylglyoxal bis (guanyl hydrazone) and related antiprotozoals on Acanthamoeba culbertsoni

Methylglyoxal bis(guanyl hydrazone) (MGBG) and the related diamidine compounds berenil and pentamidine inhibited multiplication of A. culbertsoni. The growth inhibition by MGBG (2.5 mM) in the peptone medium was accompanied by the disappearance of spermidine and a marked reduction in the level of diaminopropane. MGBG and berenil completely inhibited growth in a chemically defined medium at 1 mM and 1-2 microM concentration, respectively. However, there was no decrease in the polyamine levels in the early stages of growth inhibition by these agents. Uptake of putrescine, spermidine and spermine by A. culbertsoni has been demonstrated but addition of exogenous polyamines did not reverse the growth inhibitory action of MGBG and berenil. Inhibition of S-adenosylmethionine decarboxylase and decrease in polyamine synthesis do not seem to be the primary targets for the antiamoebic action of MGBG and berenil.     

Fourier transform Raman study of the structural specificities on the interaction between DNA and biogenic polyamines

Biogenic polyamines putrescine, spermidine, and spermine are essential molecules for proliferation in all living organisms. Direct interaction of polyamines with nucleic acids has been proposed in the past based on a series of experimental evidences, such as precipitation, thermal denaturation, or protection. However, binding between polyamines and nucleic acids is not clearly explained. Several interaction models have also been proposed, although they do not always agree with one another. In the present work, we make use of the Raman spectroscopy to extend our knowledge about polyamine-DNA interaction. Raman spectra of highly polymerized calf-thymus DNA at different polyamine concentrations, ranging from 1 to 50 mM, have been studied for putrescine, spermidine, and spermine. Both natural and heavy water were used as solvents. Difference Raman spectra have been computed by subtracting the sum of the separated component spectra from the experimental spectra of the complexes. The analysis of the Raman data has supported the existence of structural specificities in the interactions, at least under our experimental conditions. These specificities lead to preferential bindings through the DNA minor groove for putrescine and spermidine, whereas spermine binds by the major groove. On the other hand, spermine and spermidine present interstrand interactions, whereas putrescine presents intrastrand interactions in addition to exo-groove interactions by phosphate moieties.