Protein dissociation from DNA in model systems and chromatin

Shiffman, Mitchell L.; Maciewicz, Rose A.; Hu, Ali W.; Howard, J. C.; Li, Hsueh Jei

doi:10.1093/nar/5.9.3409

Cited by 10 publications

(8 citation statements)

References 29 publications

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“…CHAPS buffer (a commonly used lysis buffer for telomerase extraction) is not expected to extract TERT bound to DNA. Increasing the ionic strength of the solution by adding NaCl disrupts electrostatic DNA/protein interactions [24], thus adding salt to the CHAPS lysis buffer enables the release of DNA-binding proteins. Indeed, we observed that when pellets containing DNA and membrane debris, which had been previously washed three times with CHAPS buffer, were washed once more with CHAPS buffer supplemented with 1M NaCl (final concentration), the quantity of extracted protein increased by three fold (P<0.01), as compared to the previous CHAPS wash (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons

et al. 2015

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Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, an enzyme that elongates telomeres at the ends of chromosomes during DNA replication. Recently it was shown that TERT has additional roles in cell survival, mitochondrial function, DNA repair and Wnt signaling, all of which are unrelated to telomeres. Here we demonstrate that TERT is enriched in Purkinje neurons, but not in the granule cells of the adult mouse cerebellum. TERT immunoreactivity in Purkinje neurons is present in the nucleus, mitochondria and cytoplasm. Furthermore, TERT co-localizes with mitochondrial markers, and immunoblot analysis of protein extracts from isolated mitochondria and synaptosomes confirmed TERT localization in mitochondria. TERT expression in Purkinje neurons increased significantly in response to two stressors: a sub-lethal dose of X-ray radiation and exposure to a high glutamate concentration. While X-ray radiation increased TERT levels in the nucleus, glutamate exposure elevated TERT levels in mitochondria. Our findings suggest that in mature Purkinje neurons TERT is present both in the nucleus and in mitochondria, where it may participate in adaptive responses of the neurons to excitotoxic and radiation stress.

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Section: Resultsmentioning

confidence: 99%

Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons

et al. 2015

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“…There are differences in detail showing that base composition and sequence have some influence. By light scattering Shiffman et al (1978) found that (dG),.…”

Section: Methodsmentioning

confidence: 99%

Interactions of poly(N.epsilon.,N.epsilon.,N.epsilon.-trimethyllysine) and poly(N.delta.,N.delta.,N.delta.-trimethylornithine) with polynucleotides: salt dissociation and thermal denaturation

Granados¹,

Bello²

1980

Biochemistry

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The interaction of poly(N epsilon, N epsilon, N epsilon-trimethyl-L-lysine) ([Lys(Me3)]n) and poly(N delta, N delta, N delta-trimethyl-L-ornithine) ([Orn(Me3)]n) with polynucleotides was studied by thermal denaturation, viscosity, and dissociation by salt. The methylated polymers decrease the viscosity of DNA in proportion to the amount of bound peptide. [Lys(Me3)]n and [Orn(Me3)]n raise Tm of polynucleotides more than do (Lys)n and (Orn)n. Dissociation of the polypeptide-polynucleotide complexes with NaCl, KCl, or MgCl2 required about half the salt concentration for the methylated polymers as for the parent polymers. The effects of Tm on DNA appear to be complex and may involve differences in the hydrophobic effects, solvation, and conformational entropy. The salt dissociation data are discussed in relation t the role of histone methylation in chromatin function.

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“…CD spectra of polypeptides were measured with 1 mm cells and of polynucleotides with 1 em cells. Dissociation of polynucleotide-polypeptide complexes by salt was followed by the light scattering method of Schiffman et al (22).…”

Section: Methodsmentioning

confidence: 99%

Methylated Polyl(L-lysine): Conformational Effects and Interactions with Polynucleotides

Bello

Granados

Lewinski

et al. 1985

Journal of Biomolecular Structure and Dynamics

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Methylated lysine, arginine and histidine residues are found in a number of proteins (for example, histones, non-histone chromosomal proteins, ribosomal proteins, calmodulin, cytochrome C, etc.). We are studying the effects of methylation on the conformations of poly(lysine) and of the effects of methylation of poly(lysine) and poly(arginine) on interactions with polynucleotides. The conformational properties of epsilon-amino-methylated poly(lysine) differ from those of unmodified poly(lysine). Methylation increases resistance to thermally-induced and NaCl-induced changes in the CD spectrum. Guanidinium chloride increases (proportional to the degree of methylation) the extent of approach to the conformation in dispute as to its being a random coil or an extended helix. Methylation enhances aggregation in the helix-inducing solvent 0.5 M Ca(ClO4)2. With increasing methylation of poly(lysine), the conformation in dodecyl sulfate changes from beta, to 50% alpha, to random coil at the maximum methylation. Increasing methylation of poly(lysine) weakens the interaction with polynucleotides in respect to dissociation by salt, linearly with methyl content. Complexes of (dAdT)n.(dAdT)n with the polypeptides are increasingly stabilized to heat denaturation by progressive methylation. However, with a series of synthetic double-stranded RNA's and DNA's a more complex situation exists, Tm increasing or decreasing, depending on the base composition, sequence and type of sugar. Methylation of poly(lysine) and poly(arginine) can have opposite effects on Tm based on results with complexes with (dI)n.(dC)n. Methylated poly(lysine) affects the CD spectrum of polynucleotides, in a manner dependent on base composition and sequence. In some cases large positive or negative psi-spectra are induced, which, in the case of (dGdC)n.(dGdC)n, can be positive or negative depending on the degree of methylation of the polypeptide and the salt concentration. It is suggested that the biological effects of methylation proteins may be evoke by salt changes in the cell cycle, and that methylation can affect local interactions with nucleic acids and larger scale structure, and interactions with lipids.

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Protein dissociation from DNA in model systems and chromatin

Cited by 10 publications

References 29 publications

Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons

Excitotoxic and Radiation Stress Increase TERT Levels in the Mitochondria and Cytosol of Cerebellar Purkinje Neurons

Interactions of poly(N.epsilon.,N.epsilon.,N.epsilon.-trimethyllysine) and poly(N.delta.,N.delta.,N.delta.-trimethylornithine) with polynucleotides: salt dissociation and thermal denaturation

Methylated Polyl(L-lysine): Conformational Effects and Interactions with Polynucleotides

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