Excision of apurinic and/or apyrimidinic sites from DNA by nucleolytical enzymes from rat brain

Apurinic and/or apyrimidinic (AP) sites were excised from PM2 phage DNA by two enzymes: an AP endodeoxyribonuclease isolated from rat neocortex chromatin and a rat brain exodeoxyribonuclease, DNase B III. The resulting gap was filled with DNA polymerase beta prepared from rat liver and finally ligated by Escherichia coli DNA ligase.     

Repair of depurinated DNA with enzymes from rat liver chromatin.

DNA from T7 phage containing AP (apurinic/apyrimidinic) sites was repaired by the successive actions of three chromatin enzymes [AP endodeoxyribonuclease, DNAase IV (5'----3'-exodeoxyribonuclease) and DNA polymerase-beta] prepared from rat liver and T4-phage DNA ligase. Since DNA ligase is also found in rat liver chromatin, all the activities used for the successful repair in vitro are thus present in the chromatin of a eukaryotic cell. Our results show, in particular, that the chromatin DNAase IV is capable of excising the AP site from the DNA strand nicked by the chromatin AP endodeoxyribonuclease. We did not try to combine all the enzymes, since competition between some of them might have prevented the repair; we have, for instance, shown that DNA ligase can seal the incision 5' to the AP site made by the AP endodeoxyribonuclease. Changes in chromatin structure during repair might perhaps prevent this competition when nuclear DNA is repaired in the living cell.     

Properties of activated chromatin from rat liver shortly after partial hepatectomy

The properties of rat liver chromatin 1, 2 and 6 hours after partial hepatectomy have been studied by means of cytochemical and biochemical methods. An increase in the accessibility of DNA to low molecular weight ligands, RNA--polymerase and RNAse I and also of the distances between nucleosomes and their heterogeneity in length on electron -- microscopic photographs has been found. Analysis of the isotherms of adsorption has revealed an increase in the number of binding sites for ethidium bromide on DNA and accordingly a decrease in the extent of the filling of the template with protein in activated chromatin. Two hours after partial hepatectomy rat liver chromatin does not differ in all parameters studied from control chromatin. Limited digestion of chromatin with DNAse I almost fully eliminates the difference between the fractions of activated and control chromatin in the number of binding sites for the ligands to the fractions resistent in these conditions to nuclease. A suggestion that the changes in the properties of chromatin upon activation are due to the change in the character of chromatin proteins interaction with DNA are discussed.     

In vitro interaction of 63-nickel(II) with chromatin and DNA from rat kidney and liver nuclei

The interaction of nickel(II) with chromatin was studied in vitro and in isolated nuclei from rat liver and kidney. Nickel(II) bound to chromatin, polynucleosomes (DNA + histone octamer protein complex), and to deproteinized DNA both in intact nuclei and in vitro. The amount of nickel(II) bound depended on the concentration of nickel(II), the presence of chromosomal proteins and the binding sites on DNA which provide a stable coordination environment for nickel(II). The binding of nickel(II) to chromatin and to DNA in whole nuclei was much slower than in vitro indicating that assessibility of the DNA binding sites was influenced by the presence of the nuclear membrane, nuclear matrix and nuclear proteins and/or by the condensed nuclear structure of chromatin. Since DNA containing bound nickel(II) was isolated from chromatin, nickel(II) directly interacted with stable binding sites on the DNA molecule in chromatin. Nickel(II) was associated with the histone and non-histone nuclear proteins as well as the DNA in rat liver and kidney chromatin. Nickel(II) was found to bind to calf thymus histones in vitro. Nickel(II)-nuclear protein and -DNA interactions were investigated by gel electrophoretic analysis of in vitro incubation products. Although nickel-histone and nickel-non-histone protein interactions were completely disrupted by the electrophoretic conditions, fluorography revealed the presence of inert nickel(II)-DNA and/or nickel(II)-DNA-protein complexes.     

Developmental and regional variations in ribonucleic acid synthesis on cerebral chromatin

1. Chromatin was prepared from purified nuclei isolated from liver and cerebral regions of the rat. 2. The capacity of these preparations to promote RNA synthesis in the presence of bacterial RNA polymerase was determined. 3. The rate of RNA synthesis on chromatin was normally 12-21% of the rate observed with native DNA, but was markedly stimulated on addition of 200mm-ammonium sulphate. 4. At physiological concentrations (80mug./ml.), the brain-specific S-100 protein inhibited RNA synthesis on DNA and chromatin. 5. Cerebral chromatin from foetal and newborn animals was more active in RNA synthesis than were the analogous preparations from liver. 6. Cerebellar chromatin maintained a high rate of RNA synthesis during brain maturation. In contrast, RNA synthesis on chromatin from other brain regions and liver declined with age of the rat. 7. RNA synthesized on chromatin stimulated amino acid incorporation in an Escherichia coli ribosomal system and hybridized with homologous DNA. 8. RNA synthesized on chromatin from adult cortex or hindbrain hybridized with DNA to a greater extent than that synthesized on cerebellar chromatin. 9. The proportion of RNA formed on cerebral-cortical chromatin that hybridized with DNA increased with age of the rat. 10. The results indicate that the total amount and the types of RNA synthesized on cerebral chromatin vary regionally and during development.     

Identification of a single-stranded DNA binding protein from rat liver with high mobility group protein 1

The rat liver single-stranded DNA binding protein, S25 and HD25, isolated by differential DNA cellulose affinity chromatography was compared to the high mobility group proteins, HMG1 and HMG2, isolated from rat liver chromatin by the technique of Goodwin et al. (Goodwin, G. H., Sanders, C., and Johns, E. W. (1973) Eur. J. Biochem. 38, 14-19). Analysis of their amino acid composition, electrophoretic mobility, and tryptic peptide map reveal the identity of the single-stranded DNA binding protein with HMG1 protein, implying that the rat liver HMG1 protein becomes able both to destabilize a double helix of DNA and to stimulate homologous DNA polymerases only when rat liver cells enter a phase of DNA synthesis, possibly after a specific modification.     

Distribution of 7-methylguanine and of replication sites in the different kinetic classes of DNA from rats treated with dimethylnitrosamine.

The distribution of 7-methylguanine in the families of repetitive and unique sequences of rat liver chromatin DNA has been studied using the technique of DNA-DNA reassociation. Rats were injected with di[14C]methylnitrosamine and chromatin DNA was prepared 3 h later. The distribution of 7-methylguanine was found to be random between these classes of DNA. We have also studied chromatin DNA from rats treated with unlabelled DMN plus [3H]thymidine in this way, in order to find if DMN affects DNA synthesis within any one kinetic class. Our results suggest that there is no difference in the extent of synthesis between these classes.     

Nucleosome structure in Aspergillus nidulans

The structure of chromatin from Aspergillus nidulans was studied using micrococcal nuclease and DNAase I. Limited digestion with micrococcal nuclease revealed a nucleosomal repeat of 154 base pairs for Aspergillus and 198 base pairs for rat liver. With more extensive digestion, both types of chromatin gave a similar quasi-limit product with a prominent fragment at 140 base pairs. The similarity of the two limit digests suggests that the structure of the 140 base pair nucleosome core is conserved. This implies that the difference in nucleosome repeat lengths between Aspergillus and rat liver is caused by a difference in the length of the DNA between two nucleosome cores. Digestion of Aspergillus chromatin with DNAase I produced a pattern of single-stranded fragments at intervals of 10 bases which was similar to that produced from rat liver chromatin.     

DNA polymerase beta from the rat liver. Isolation, properties and inhibitory analysis of a homogeneous preparation

A simple and reproducible purification procedure of homogeneous DNA polymerase beta from rat liver is developed, including sedimentation and saline extraction of rat liver chromatin, chromatography of the extract on DEAE-cellulose, phosphocellulose, Gel Blue A, and DNA sepharose. The purified enzyme isolated with the 8.4% yield proved to be a homogeneous protein with m.w. 38-40 kDa, specific activity 31 units/g, pI 8.6-8.9. Incorporation of [3H]TTP into activated DNA catalysed by DNA polymerase beta was strongly inhibited by dNTP (3'NH2), ddTTP, dNTP (3'F) and slightly inhibited by aCTP and aNTP (3'NH2).     

Restoration of the primer activity of gamma-irradiated DNA by nucleases from rat liver chromatin

gamma-Irradiation of DNA results in a several-fold decrease of its primer activity measured as one substrate synthesis catalyzed by DNA polymerase beta. However, the combined treatment of injured DNA with 3'----5' exonuclease and endonuclease I from rat liver chromatin almost normalizes primer activity of DNA. Therefore the above-mentioned nucleases are capable of excising the gamma-injured nucleotides from 3'-OH ends of DNA.     

Stimulation of DNA synthesis in isolated nuclei of rat liver cells by neutral Mn-dependent DNase of chromatin

It was demonstrated that neutral Mn-dependent DNAase from rat liver chromatin stimulates the incorporation of labeled precursors of DNA into high molecular weight fractions of isolated nuclear DNA. The effects of DNA-polymerase inhibitors and the properties of DNA synthesis products suggest that neutral Mn-dependent DNAase can induce replicative synthesis of DNA in the nuclei of normal and regenerating rat liver.     

Mode of inhibition of DNA synthesis induced by adenosine diphosphoribosylation of nuclear protein

DNA obtained after complete removal of the proteins from NAD-treated rat liver chromatin possessed template capacity equivalent to that obtained from untreated chromatin. The stepwise extraction of proteins from chromatin affected a gradual removal of labeled ADPR and a parallel decrease of the inhibition. The results of the present study suggest that the inhibition of the template capacity for DNA synthesis following preincubation of chromatin with NAD is dependent upon ADP-ribosylation of the associated proteins. The template capacity of chromatin and nucleoprotein complex (Weiss preparation) for DNA synthesis was inhibited, whereas the capacity for RNA synthesis was apparently not affected.     

Studies on the Action of the Nuclear Factor Promoting Actinomycin D-Binding Capacity of Chromatin

It is well known that actinomycin D binds to C-G pairs of DNA. The amount of actinomycin D bound to chromatin thus depends directly on the demasked sites of chromatin DNA. The actinomycin D binding of rat liver chromatin, obtained by the method of Dingman and Sporn, was studied in the presence and absence of liver and kidney nuclear extracts (NE). The actinomycin D binding of liver chromatin increases greatly under the action of liver nuclear extract. No changes occur in liver chromatin actinomycin D binding capacity after the action of kidney NE. The removal of protein or RNA from liver NE removes its ability to change the actinomycin D binding capacity of the liver chromatin. According to the obtained results it may be assumed that the nuclear extract contains the factor which plays a role in controlling cell differentiation.     

Localization of the phosphoester bond hydrolyzed by the major apurinic/apyrmidinic endodeoxyribonuclease from rat-liver chromatin

The major apurinic/apyrimidinic (AP) endodeoxyribonuclease from rat liver chromatin, an enzyme specific for AP sites in DNA, cleaves the phosphodiester bridge which is the immediate neighbour of the AP site on its 5' side leaving 3'-hydroxyl and 5'-phosphate ends. In contrast with Escherichia coli endonuclease VI, this chromatin enzyme is inactive on reduced AP sites.     

Studies on the action of the nuclear factor promoting actinomycin D-binding capacity of chromatin

It is well known that actinomycin D binds to C-G pairs of DNA. The amount of actinomycin D bound to chromatin thus depends directly on the demasked sites of chromatin DNA. The actinomycin D binding of rat liver chromatin, obtained by the method of Dingman and Sporn, was studied in the presence and absence of liver and kidney nuclear extracts (NE). The actinomycin D binding of liver chromatin increases greatly under the action of liver nuclear extract. No changes occur in liver chromatin actinomycin D binding capacity after the action of kidney NE. The removal of protein or RNA from liver NE removes its ability to change the actinomycin D binding capacity of the liver chromatin. According to the obtained results it may be assumed that the nuclear extract contains the factor which plays a role in controlling cell differentiation.     

Immunofractionation of chromatin regions associated with histone H1o

Two monoclonal antibodies, which were elicited against histone H5, bind to purified rat liver chromatin and to rat liver H1o but not to rat liver H1. The monoclonal antibodies were immobilized on CNBr-Sepharose and the resulting immunoaffinity column was used to fractionate rat liver oligonucleosomes. Enzyme-linked immunoabsorbant assay (ELISA) and immunoblotting experiments indicate that the nucleosomes bound to the column were tenfold enriched in their content of H1o. Oligonucleosomes, prepared from the livers of either untreated or 3-methylcholanthrene-treated adult rats, were fractionated on the anti-H1o affinity column. The DNA purified from the unfractionated nucleosomes, from the unbound nucleosomes and from the nucleosomes which were bound to the column was examined with various 32P-labeled probes. A slight enrichment in H1o was detected in the coding region of the rat albumin gene. In contrast DNA which was bound to the column was significantly depleted in sequences hybridizing with total cellular RNA (which contains mostly ribosomal RNA) and with sequences hybridizing to the 3'-terminal region of a cytochrome P-450 gene, which is inducible by the chemical carcinogen 3-methylcholanthrene, regardless of whether isolated from control or from carcinogen-treated rat livers. Our experiments clearly demonstrate that chromatin can be efficiently immunofractionated. The results suggest that the H1o content of chromatin regions containing genes which are constitutively transcribed is not necessarily different from that of regions containing non-transcribed genes and that highly inducible genes may be segregated into chromatin regions which are depleted of H1o.     

Interaction of the DNA untwisting enzyme with the SV40 nucleoprotein complex.

The SV40 nucleoprotein complex which was isolated from infected CV-1 cells did not possess an active DNA untwisting enzyme. The superhelix density of the DNA in the chromatin complex was unchanged after treatment with purified rat liver DNA untwisting enzyme. However, in the presence of ethidium bromide (1 microgram/ml) the superhelix density was changed. Moreover, the nicked intermediate in the DNA untwisting reaction could be detected using the chromatin DNA as a substrate. These results show that the DNA in the SV40 chromatin which is accessible to the DNA untwisting enzyme is under no topological strain.     

An electrophoretic comparison of non-histone proteins from rat liver total chromatin and chromatin depleted of 0.35 M NaCl soluble proteins

• 1.1. A procedure of isolation of non-histone proteins from rat liver chromatin in mild conditions provided 3 groups of these proteins, i.e. NHCP1, NHCP2 and NHCP3.
• 2.2. The investigated proteins are devoid of DNA and revealed various influences on RNA synthesis in vitro.
• 3.3. The extraction of rat liver chromatin with 0.35 M NaCl (pH 7.5) removed about 30% of examined proteins. Electrophoretic patterns of 3 groups of non-histone proteins from total chromatin and chromatin depleted of 0.35 M NaCl soluble proteins are compared.