Accessibility of DNA in Chromatin to DNA Polymerase and RNA Polymerase

Abstract

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

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

“…Other evidence for this viewpoint was provided by Stollar (18), who found that in native nucleoprotein (extracted chromatin) 1-5% of the DNA was available to react with antibody to DNA compared to about 20% of the DNA in dissociated-reassociated nucleo-protein. This finding is consistent with our observations that DNA in chromatin in the nucleus is only slightly accessible to deoxyribonuclease (2) and exogenous DNA arid RNA polymerases (13).…”

“…Incubations and assays with E. coli DNA polymerase at pH 7.4 and RNA polymerase at pH 7.9 have been described (13). Evidence that the nuclear membrane does not affect accessibility to these enzymes has also been l)resented (1, 13).…”

“…No significant differences in either Additions and assays were performed as indicated in the text and in Table 1. Exogenous E. coli RNA polymerase was used in these assays (13). Incubations were for 20 min at a DNA concentration of 200 ,ug/ml.…”

Enhancement of DNA Synthesis in a Mammalian Cell-Free System by Trypsin Treatment

“…Cells were either removed by a brief trypsin treatment or else scraped off in isolation buffer with a rubber policeman. In either case, the cells were washed once in 4 ml of isolation buffer (5 X 106 cells per ml) at 40 and then pelleted by centrifugation at 800 X g for 5 min at 4°. The pellet was then resuspended in isolation buffer at approximately 2 X 107 cells per ml.…”

“…Recently it has been shown that a short chain of RNA is associated with the nascent DNA, and it has been suggested that this RNA must be synthesized before the DNA can be replicated (3). Silverman and Mirsky (4) have shown that exogenous DNA polymerase and RNA polymerase both have much less access to the DNA in isolated nuclei or chromatin than in isolated DNA.…”