An auxiliary protein for DNA polymerase-delta from fetal calf thymus

An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.     

Effect of denaturation of DNA template on activity of RNA polymerase of class B

The effect of the denaturation of homologous and calf thymus DNA on the RNA polymerase B activity purified from rat liver and spleen and Ehrlich ascites cells, was investigated in presence of either Mn2+ or Mg2+ and in presence or absence of alpha-amanitin. On the basis of the results here reported, we suggest: 1) denatured DNA is more effective than native as template for polymerase B; 2) denatured DNA template and cations might play a role in determining the extent of the reaction alpha-amanitin-polymerase B.     

Osmium-induced alteration in DNA structure

In the presence of pyridine and other ligands osmium tetroxide binds covalently to pyrimidine bases in DNA. Properties of osmium-modified native and denatured calf thymus DNA, and plasmid Co1E1 DNA were investigated by means of differential pulse polarography, absorption spectrophotometry, circular dichroism, agarose gel electrophoresis, and nuclease S1 digestion. A great difference in the reaction kinetics of native and denatured DNAs with osmium, pyridine was observed. On the ground of the slow stepwise reaction kinetics of native DNA in the initial stage of its modification by osmium it has been suggested that the primary reaction sites do not include bases contained in the intact double helix. Osmium binding to sporadic primary reaction sites (represented e.g. by bases in the vicinity of a single-strand break) in native calf thymus DNA resulted in local changes in DNA conformation limited to a close neighbourhood of the binding site. At higher osmium/nucleotide ratios disordering of the DNA structure over a region extending beyond the immediate binding site was observed. With denatured DNA the same type of structure disordering was detected already in the initial stage of the reaction at osmium/nucleotide ratios as low as 0.01. Osmium binding to the supercoiled Co1E1 DNA resulted in its relaxation without nicking and it increased its sensitivity to linearization by cleavage with nuclease S1. The behaviour of Co1E1 DNA has been explained by the formation of a denatured region in the molecule (accompanied by a coupled loss of duplex and superhelical turns). It has been suggested that osmium can be used to label and to visualize distorted regions in the DNA double helix.     

Evidence implying DNA polymerase beta function in excision repair.

Comparison was made of the ability of calf thymus DNA polymerases alpha and beta to replicate the following templates: native E. coli CR-34 DNA (T-DNA), calf thymus DNA activated by DNase I (act.DNA), BU-DNA (from E. coli CR-34 cells cultured on BUdR-containing medium) with damages resulting from incomplete excision repair, as well as thermally denatured act.DNA and BU-DNA (s.s.act.DNA and s.s.BU-DNA). 3H-TTP incorporation during extensive replication of act.DNA was similar for both enzymes, being, as expected, 40 times higher than for T-DNA. Likewise, the differences in the yield of the s.s.act.DNA or s.s.BU-DNA replication between both enzymes were negligible. In contrast, damaged native DNA was 6 - 30 times more extensively replicated by DNA polymerase beta than alpha. We propose that this is due to the greater ability of DNA polymerase beta compared with alpha to replicate single-stranded gaps, the presence of which is more likely in damaged BU-DNA than in T-DNA and act.DNA.     

Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis.

A method is described to detect DNA polymerases and nucleases in homogeneous or crude enzyme preparations after electrophoresis in SDS-polyacrylamide gels(2) containing the appropriate template or substrate. DNA polymerases are electrophoresed in a gel containing gapped calf thymus DNA and after a renaturation treatment, the gel is incubated in a reaction mixture in which one deoxyribonucleoside triphosphate is [alpha-32P]-labelled. Incorporation of radioactivity into DNA is detected at the vicinity of the polymerase band by autoradiography. An associated nuclease activity can be measured after electrophoresis in a gel containing 32P-labelled gapped DNA, when nucleolytic digestion is seen as a clear band in the resulting autoradiogram. The gels can subsequently be stained with Coomassie blue to establish identical molecular weights of polymerase, nuclease and protein bands. Applications of this technique are discussed.     

Inhibition of chicken myeloblastosis RNA polymerase II activity by adriamycin.

In vitro RNA synthesis by isolated RNA polymerase II of chicken myeloblastosis cells was shown to be highly sensitive to adriamycin inhibition. The template activity of the single-stranded DNA, purified by chromatography of denatured calf thymus DNA through hydroxylapatite columns, was found to be equally as sensitive to the inhibition as denatured calf thymus DNA. However, contrary to denatured DNA, the single-stranded DNA thus purified showed no significant binding to adriamycin as analyzed by cosedimentation of the drug and DNA through a sucrose gradient. This indicated that inhibition of RNA synthesis on a single-stranded DNA template might involve a mechanism other than DNA intercalation. Kinetic studies of the inhibition showed that the inhibition of RNA synthesis by adriamycin could not be reversed by increasing the concentrations of RNA polymerase and four nucleoside triphosphates, but it could be reversed by increasing DNA concentrations. Analysis of the size of RNA synthesized indicated that the ultimate size of the product RNA was not altered by adriamycin, suggesting that the drug may inhibit RNA synthesis by reducing RNA chain initiation.     

EFFECT OF ACIDIC DEOXYRIBONUCLEOPROTEINS FROM BRAIN ON DNA SYNTHESIS IN VITRO

Chromatin acidic proteins extracted from rat brain stimulate in uitro synthesis of DNA with either brain chromatin. native or denatured DNA as template. The stimulation is due to the presence of nuclease activity within these proteins which increases the priming activity of DNA.     

The DNase activity of an endoplasmic reticulum nuclease and its effect on DNA synthesis in vitro

An endoplasmic reticulum nuclease which was isolated previously in this laboratory from rat liver ( Kouidou et al. (1981) Eur.J. Bioch . 120, 9-14) was found to degrade linear and circular single stranded DNA but not double stranded DNA. The DNA fragments resulting from this cleavage were longer than 20 nucleotides. In addition the nuclease was found to improve the efficiency of DNA template used by DNA polymerase I in DNA synthesis in vitro. The results were the same whether incubation of the template with the nuclease was prior to addition of DNA polymerase I or simultaneously with polymerization. When nuclease was added after the completion of polymerization by DNA polymerase I it was ineffective unless the product was denatured. These data further corroborate the observation that double stranded DNA is not cleaved by this enzyme.     

Isolation of foldback DNA utilizing nuclease S1 digestion in aqueous dioxane.

An improved method for the isolation of the inverted repetitive (foldback) sequences present in mammalian DNAs is described. It makes use of the new observation that nuclease S1 digestion of denatured DNA occurred at a faster rate and was more extensive in medium containing dioxane. The temperature-absorbance characteristics of nuclease S1-resistant DNA were systematically studied as a function of the temperature employed during the step of enzymic hydrolysis. Specimens of human placental and calf thymus DNA which had been denatured and renatured to C₀t ≤ 10^?3 mol s liter^?1 were used as substrates. Foldback DNA was isolated from the enzymic digests by means of hydroxylapatite chromatography. Temperature-absorbance studies showed the enzyme-resistant DNA had a high degree of thermal stability; the hyperchromic rises equaled those obtained in the native speciments. The amount of foldback DNA which could be obtained was not influenced by the fragment size of the starting material, above a certain molecular weight range. Foldback DNA represented about 4% of the human genome and at least 5% of the bovine genome. The size distributions of these strands were studied by means of polyacrylamide gel electrophoresis.     

Influence of template inactivators on the binding of DNA polymerase to DNA

The agents daunomycin, ethidium bromide, distamycin A and cytochrome c inhibit DNA dependent DNA polymerase I (E. coli) reaction competitively to DNA. The influence of these template inactivators on the binding of DNA polymerase to native as well as denatured DNA has been determined by affinity chromatography. Cytochrome c blocks the binding of the enzyme to double-stranded and to single-stranded DNA Sepharose. In contrast to these results daunomycin, ethidium bromide or distamycin A reduce the binding affinity only with denatured DNA Sepharose as matrix. These data are discussed with respect to the modification by template inactivators of the affinity of DNA to the different binding sites of the DNA polymerase.     

Apparent stimulation of calf thymus DNA polymerase alpha by ATP.

The mechanism by which millimolar concentrations of ATP stimulate the activity and increase the processivity of calf thymus DNA polymerase alpha has been investigated with poly(dA)/oligo(dT) as template/primer to eliminate possible effects due to primer synthesis. The effect of ATP on the rate of DNA synthesis with this template/primer was found to be dependent upon whether or not the ATP was neutralized and the species of buffer used in the reaction. The present studies suggest that ATP stimulation of calf thymus DNA polymerase can be attributed to changes in the pH of the reaction mixture, a shift in the magnesium ion optimum, or both. Furthermore, effects of ATP on the processivity of DNA polymerase alpha could be mimicked by lowering the pH of the reaction mixture.     

INCORPORATION OF NUCLEOTIDES INTO NUCLEI OF FIXED CELLS BY DNA POLYMERASE

The enzyme calf thymus polymerase requires denatured or single-stranded DNA as a primer for DNA synthesis and is inactive on native DNA preparations. The enzyme and tritium-labeled deoxyribonucleoside triphosphates were incubated with alcohol-fixed and Carnoy-fixed tissue preparations to see if primer DNA could be found in several types of cells undergoing DNA synthesis. In all cases, low-pH controls were prepared for comparison. Priming activity was not found in nuclei that had been fixed in alcohol. Priming activity was found in cell nuclei that had been fixed with an acid fixative or had been treated at a low pH prior to treatment with the enzyme reaction mixture.     

DNA primase associated with 10 S DNA polymerase alpha from calf thymus

Among multiple subspecies of DNA polymerase alpha of calf thymus, only 10 S DNA polymerase alpha had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase alpha through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase alpha. These results indicate that the primase is tightly bound to 10 S DNA polymerase alpha. The RNA polymerizing activity was resistant to alpha-amanitin, required high concentration of all four ribonucleoside triphosphates (800 microM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase alpha because it was strongly inhibited by araCTP, resistant to d2TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.     

DNA sequence directs placement of histone cores on restriction fragments during nucleosome formation.

Restriction fragments, 203 and 144 base pairs in length, bearing the Escherichia coli lac control region have been reconstituted with the core histones from calf thymus to form nucleosomes. By several criteria the reconstituted nucleosomes are similar to native nucleosomes obtained by micrococcal nuclease digestion of calf thymus nuclei. However, sensitive nuclease digestion studies reveal subtle and important differences between native monosomes and the lac reconstitutes. Each reconstitute consists mainly of nucleosomes containing histone cores placed nonrandomly with respect to the DNA sequence. The shorter reconstitute forms asymmetric nucleosomes as evidenced by the DNase I digestion pattern. Exonuclease III digestion followed by 5'-end analysis of the larger reconstitute suggests that, of the many possible arrangements of histone core with DNA sequence, only two are highly favored.     

Single strand DNA binding of simian virus 40 tumor antigen.

Simian virus 40 T antigen binds to both single and double strand DNA. The single and double strand DNA binding activity of crude T antigen preparations was evaluated by chromatography of the antigen on DNA-cellulose columns. Crude T antigen was retained on both native and denatured DNA-cellulos columns and was eluted from both columns under similar conditions. The interaction of highly purified T antigen with single and double strand DNA was evaluated by competition experiments using a DNA filter binding assay. These experiments showed that T antigen binds preferentially to single strand calf thymus DNA by more than an order of magnitude when compared to double strand calf thymus DNA.     

Specific limited cleavage of bihelical deoxyribonucleic acid by wheat seedling nuclease.

Wheat seedling nuclease catalyzes the hydrolysis of intact, bihelical viral DNA or high molecular weight, native Escherichia coli DNA to produce limit polymers which are resistant to further hydrolysis by additional enzyme. These limit products are double-stranded polymers free of single strand interruptions and are terminated at their 5' ends with equal amounts of either deoxycytidylate or deoxyguanylate residues. The average size of the duplex limit products, as determined by (a) alkaline and neutral sucrose gradient sedimentation, (b) viscometric determination of molecular weight, and (c) 5'-end labeling, varies from 2 to 4 times 10-6 depending on the source of the DNA. The involvement of regions rich in adenine-thymine base pairs at the sites of cleavage of the DNA molecule is suggested by the following experimental results: (a) the copolymeric duplex, poly(dA-dt) is hydrolyzed at a rate comparable to that found for denatured calf thymus DNA, a rate which is several orders of magnitude faster than that at which native calf thymus DNA is hydrolyzed; (b) lambda DNA, which contains an adenine-thymine-rich region near its center, is rapidly cleaved to yield two fragments of similar size; (c) the rate of hydrolysis of native DNA is increased approximately 14-fold by increasing the reaction temperature from 20 degrees to 30 degrees.