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

Abstract

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 ...

Lysine-rich histones and the selective digestion of the globin gene in avian red blood cells

“…Since we have ruled out any irreversible effects on DNase I digestion kinetics of the low-pH salt extraction procedure used to remove histones HI and H5 from erythroid nuclei, our results suggest that the lysine-rich histones HI and H5 have a marked effect on DNase I digestion kinetics. This is consistent with the findings of Silverman and Mirsky (1973) that removal of H1 histone from calf thymus nuclei greatly increases the rate of digestion with DNase I.…”

Properties of Basic Amino‐Acid Residues

“…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.…”

Mammalian chromatin substructure studies with the calcium–magnesium endonuclease and two-dimensional polyacrylamide-gel electrophoresis

“…The chromatin of mammalian cell nuclei consists of DNA complexed with basic and acidic proteins which have long been suspected to be involved in control of the expression of the genetic material. Previous studies have shown that the nuclear DNA is quite extensively covered by the nuclear proteins and much of the genome is thus inaccessible to the actions of various enzymes, such as deoxyribonuclease (Pederson, 1972;Billings & Bonner, 1972;, RNA polymerase (Bonner & Huang, 1963;Silverman & Mirsky, 1973;Paul & Gilmour, 1969) and DNA polymerase (Silverman & Mirsky, 1973;Wang, 1969). Removal of the chromatin proteins removes the inhibiting influence and allows the enzymes to act on the DNA (Billings & Bonner, 1972;Silverman & Mirsky, 1973;Wang, 1969).…”

“…Previous studies have shown that the nuclear DNA is quite extensively covered by the nuclear proteins and much of the genome is thus inaccessible to the actions of various enzymes, such as deoxyribonuclease (Pederson, 1972;Billings & Bonner, 1972;, RNA polymerase (Bonner & Huang, 1963;Silverman & Mirsky, 1973;Paul & Gilmour, 1969) and DNA polymerase (Silverman & Mirsky, 1973;Wang, 1969). Removal of the chromatin proteins removes the inhibiting influence and allows the enzymes to act on the DNA (Billings & Bonner, 1972;Silverman & Mirsky, 1973;Wang, 1969). There is, however, some argument about the degree to which the DNA is covered by the histones and the degree to which the covering proteins interfere with the approach ofprobe molecules (e.g.…”