Blocking by Histones of Accessibility to DNA in Chromatin

Mirsky, A. E.; Silverman, B. D.

doi:10.1073/pnas.69.8.2115

Cited by 32 publications

(8 citation statements)

References 21 publications

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“…However, with RNA polymerase, neither histone reduced template activity to that of intact nuclei, as was found with lysine-rich histone in DNA polymerase assays. These results, coupled with earlier observations (1,6,7), suggest that lysine-rich histone plays a unique role in regulating replication of DNA.…”

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confidence: 67%

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Addition of Histones to Histone-Depleted Nuclei: Effect on Template Activity Toward DNA and RNA Polymerases

Silverman

Mirsky

1973

Proc. Natl. Acad. Sci. U.S.A.

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The ability of histones to block the accessibility of DNA in chromatin to DNA and RNA polymerases was measured by addition of lysine-rich or arginine-rich histones to nuclei selectively depleted of these histones. By this procedure nuclei were obtained in which all of the original lysine-rich histone in the chromatin was replaced by arginine-rich histone. Conversely in other nuclei, additional lysine-rich histone replaced some of the endogenous arginine-rich histone. Lysine-rich histone was much more effective than arginine-rich histone in blocking accessibility to DNA polymerase. Both classes of histone inhibited template activity toward RNA polymerase to a similar extent.In addition to lysine-rich histone and total arginine-rich histone, phosphorylated lysine-rich histone, two fragments of lysine-rich histone produced by cleavage with N-bromosuccinamide, and fractions IIB and IV of arginine-rich histone were added to histone-depleted nuclei. With both DNA and RNA polymerases as probes, no differences in inhibition of template activity were found when native Jysine-rich histone was compared to phosphorylated lysine-rich histone. Similarly, fractions IIB and IV were indistinguishable from total arginine-rich histone. On a molar basis, the carboxyl fragment of lysinerich histone was as effective as intact lysine-rich histone only when the amino fragment was added to it. By itself, the amino portion of lysine-rich histone was without inhibitory effect in the RNA polymerase assay and resulted in only slight inhibition of template activity toward DNA polymerase.The previous paper in our studies of the accessibility of DNA in chromatin to DNA and RNA polymerases (1) described the effects of selective extraction of histones from chromatin. We found that removing lysine-rich histone greatly increased accessibility to DNA polymerase whereas extracting argininerich histone had much less of an effect. However, when RNA polymerase was used as a probe of chromatin structure, although removal of lysine-rich histone increased template activity more than removal of arginine-rich histone, neither class of histones influenced accessibility to RNA polymerase to the degree seen with DNA polymerase. We now report experiments that complement and confirm the preceding data.The experiments reported here used chromatin in the nucleus rather than solubilized extracted chromatin, since the process of solubilizing chromatin can be expected to alter some of its native properties. We have commented upon this before (1, 2) and noted the apparent difference in template activity toward RNA polymerase resulting from extraction of lysine-rich histone from chromatin in nuclei compared to extracted chromatin (1). Therefore, experiments using isolated nuclei, thus preserving the organization of the chromatin, 2637are more likely to reflect conditions in vivo than those using solubilized extracted chromatin. In this context it is important to note that in the nucleus lysine-rich histone can alter the fine structure of chromatin; its removal ...

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confidence: 67%

“…MATERIALS AND METHODS Intact nuclei, nuclei from which lysine-rich histone was extracted by 0.10 M citric acid-0.12 M NaCl, and nuclei from which arginine-rich histone was extracted by alcohol-HCl were prepared as described (3,6). Preparation of lysine-rich histone, arginine-rich histone,.…”

mentioning

confidence: 99%

Addition of Histones to Histone-Depleted Nuclei: Effect on Template Activity Toward DNA and RNA Polymerases

Silverman

Mirsky

1973

Proc. Natl. Acad. Sci. U.S.A.

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“…In polyarginine complexes only a fraction of the side chains is effective, whereas in polylysine complexes all residues can be occupied by phosphates. Similar results have been obtained with lysine-rich and arginine-rich histones from studying the accessibility of DNase to DNA and chromatin [32,33] and of DNA polymerase to DNA in chromatin [34]. Lysine-rich histones block accessibility more effectively than arginine-rich histones.…”

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confidence: 70%

Properties of Basic Amino‐Acid Residues

Wagner

Arfmann

1974

European Journal of Biochemistry

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The interaction of four basic polyamino acids, poly(L-lysine), poly(L-ornithine), poly(L-arginine) and poly(L-homoarginine) with nucleotides was investigated using equilibrium dialysis and circular dichroism. Polyhomoarginine was prepared by guanidization of polylysine. The interaction of 5'-GMP exhibited equal values for the binding cooperativity in all cases, indicating that nucleotidenucleotide interaction, i.e. the stacking tendency of the guanine base, was the origin of this affinity. The affinity parameters of 5'-GMP and other nucleotides directed towards the basic polypeptides, however, decrease in the series: polyarginine > polyhomoarginine > polyornithine > polylysine.This indicates that the ion bond between guanidinium and phosphate is stronger than that between ammonium and phosphate, and that decreasing the length of the poly(amino acid) side chains by one CH2 group results in an increase of affinity. The binding of 3'-GMP and 5'-GMP by polylysine and polyornithine induces a strong negative circular dichroism band in the absorption region of the base and in the case of polylysine also changes at shorter wavelength. Temperature-dependent measurements of these circular dichroic spectra revealed that polylysine fits best to and forms the most stable complexes with helical arrangements of bound GMP molecules.Protein-nucleic acid interactions include long-range electrostatic contributions which originate in the nucleic acid phosphates and the basic amino-acid residues of proteins, such as lysine and arginine. The ionic bond is thought not to exert much specificity. However, the chemical features of the side chains of the two basic amino acids are different enough such that one would except some specific influence on proteinnucleic acid recognition processes. Investigations in this field are chiefly those dealing with polymer-polymer interaction, for example studies of precipitation and melting of DNA * basic poly(amino acid) complexes and observations of spectral features mainly by circular dichroism (for review see [l]).There are also investigations on monomer-polymer interaction involving the binding of nucleotides by basic poly(amino acids), like polylysine and polyarginine [2-91. These offer some advantages over the polymer -polymer interaction mentioned above although they are dealing with some different aspects of the interaction. The former allows the direct determination of affinity data. In the present work comparative Part of this work was presented at the first European Biophysics Congress, Baden near Vienna, 1971. Abbreviation. CD, circular dichroic.experiments have been performed with poly(L-lysine), poly(L-ornithine), poly(L-arginine) and poly(L-homoarginine), using equilibrium dialysis and circular dichroism, in order to elucidate the influence of the nature of basic amino-acid side chains on the interaction with nucleotides. MATERIALS AND METHODSNucleotides were purchased from Boehringer Mannheim GmbH (Mannheim, Germany), poly(L-arginine) 0.5 HzS04, poly(L-lysine) HBr and poly(L-ornithine)...

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“…It should be noted that the accessibility of DNA in chromatin to DNase is also the same in liver nuclei as it is in thymus nuclei (30). The marked difference between the polymerases and DNase in their action on chromatin is that the latter at a high concentration of enzyme and in the course of time finally digests practically all of the DNA (5).…”

Section: Discussionmentioning

confidence: 99%

Accessibility of DNA in Chromatin to DNA Polymerase and RNA Polymerase

Silverman¹,

Mirsky²

1973

Proc. Natl. Acad. Sci. U.S.A.

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The accessibility of DNA in chromatin to both exogenous DNA polymerase and RNA polymerase is slight when compared to isolated DNA. DNA in extracted chromatin is somewhat more accessible to these enzymes than is DNA in the chromatin of isolated nuclei; and the DNA template of chromatin is more accessible to DNA polymerase than to RNA polymerase. In these experiments we have given much attention to the technique of scintillation counting, since artifacts arising in this procedure can lead to erroneous conclusions.Since DNA in chromatin is complexed with proteins, accessibility to DNA by enzymes and other substances is restricted. The role of histones in restricting access to DNA in chromatin was first observed by staining with basic dyes. Staining of the cell nucleus led Flemming (1) We report in this paper determinations of the accessibility of DNA in chromatin to exogenous DNA polymerase and DNA-dependent RNA polymerase. In our experiments we have studied chromatin in isolated nuclei and also in extracted chromatin (deoxyribonucleoprotein, DNP). Most of the previous work in this field has been on DNP, in which it is possible to replace certain components of chromatin, but the purpose of all these investigations is, of course, to understand chromatin as it is in the nucleus. In the experiments we are now reporting we have compared the activities of both exogenous DNA and RNA polymerases on free DNA and on DNA in both thymus nuclei and extracted chromatin. The comparison shows clearly that only a very small fraction of the DNA in a nucleus is accessible to either DNA or RNA polymerase; DNA in DNP is slightly more accessible. We have used exogenous polymerase in these experiments so that availability of the DNA template should not be obscured by enzymatic variability. It will be seen that we have given much attention to the technique of scintillation counting, for a mistake in this procedure can lead to erroneous conclusions. MATERIALS AND METHODS MaterialsThe preparation of nuclei isolated from calf thymus (6) and liver (7) in 0.25 M sucrose-3 mM CaC12 has been described. DNP (extracted chromatin) was prepared from thymus nuclei by the method of Paul and Gilmour (5, 8

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Blocking by Histones of Accessibility to DNA in Chromatin

Cited by 32 publications

References 21 publications

Addition of Histones to Histone-Depleted Nuclei: Effect on Template Activity Toward DNA and RNA Polymerases

Addition of Histones to Histone-Depleted Nuclei: Effect on Template Activity Toward DNA and RNA Polymerases

Properties of Basic Amino‐Acid Residues

Accessibility of DNA in Chromatin to DNA Polymerase and RNA Polymerase

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