Purification and characterization of 3-methyladenine-DNA glycosylase from calf thymus

The 3-methyladenine-DNA glycosylase from calf thymus has been purified and characterized. Two species of Mr = 42,000 and 27,000 +/- 5% and Stokes radius of 27.5 and 22.4 A, respectively, were found. Only the lower molecular weight species were present in the nucleus; it was bound to chromatin and could be dissociated in the presence of 0.25 M KCl. The enzymatic properties of the two species appeared to be identical. Both enzyme species released 3-methyladenine, 7-methylguanine, and 3-methylguanine, listed in the order of decreasing activity. The chromatin-associated enzyme was purified to apparent homogeneity and found to be a basic protein having a pI greater than 9. It was completely inhibited by p-hydroxymercuribenzoate, but this inhibition could be fully reversed by addition of excess 2-mercaptoethanol. Kinetic studies, heat inactivation, and inhibition experiments demonstrated that the 3-methyladenine and 7-methylguanine releasing activities were located on the same protein molecule. The enzymes showed no activity on methylated single-stranded DNA. No product inhibition was observed for any of the enzyme species, and the enzyme activity was optimal when the incubation was performed in the presence of 50 mM NaCl or KCl at pH values between 8 and 9.     

Phylogenetic evidence of a role for 5-hydroxymethyluracil-DNA glycosylase in the maintenance of 5-methylcytosine in DNA.

5-Hydroxymethyluracil (HmUra) is formed in DNA as a product of oxidative attack on the methyl group of thymine. It is also the product of the deamination of 5-hydroxymethylcytosine (HmCyt) which may be formed via oxidation of 5-methylcytosine (MeCyt). HmUra is removed from DNA by a DNA glycosylase which, together with HmCyt-DNA glycosylase, is unique among DNA repair enzymes in being present in mammalian cells but absent from bacteria and yeast. We found HmUra-DNA glycosylase activity in a wide variety of vertebrate and invertebrate animals (except Drosophila) and in protozoans. In most vertebrate organisms the highest specific activity was in nervous and immune system tissue. The phylogenetic distribution of HmUra-DNA glycosylase correlates with the presence of 5-methylcytosine (MeCyt) as a regulator of gene expression. This distribution of activity supports the contention that HmUra-DNA glycosylase aids in the maintenance of methylated sites in DNA.     

Purification and characterization of delta helicase from fetal calf thymus.

A DNA helicase (delta helicase) which partially copurifies with DNA polymerase delta has been highly purified from fetal calf thymus. delta helicase differs in physical and enzymatic properties from other eukaryotic DNA helicases described thus far. The enzyme has an apparent mass of 57 kDa by gel filtration and is associated with polypeptides of 56 and 52 kDa by SDS-polyacrylamide gel electrophoresis. Photo-cross-linking of the purified enzyme with [alpha-32P]ATP resulted in labeling of a polypeptide of approximately 58 kDa, suggesting that the active site is present on the larger polypeptide. Unwinding of a partial duplex requires a nucleoside triphosphate which can be either ATP or dATP but not a nonhydrolyzable analogue of ATP. Other ribo- and deoxyribonucleoside triphosphates have little or no activity as cofactors. delta helicase also has DNA-dependent ATPase activity which has a relatively low Km for ATP (40 microM). delta helicase binds to single-stranded DNA but has little or no affinity for double-stranded DNA or single-stranded RNA. Similar to replicative DNA helicases from prokaryotes and the herpes simplex virus type 1 helicase-primase, delta helicase translocates in the 5'-3' direction along the strand to which it is bound and preferentially unwinds DNA substrates with a forklike structure.     

Purification to homogeneity and characterization of a redoxyendonuclease from calf thymus.

A redoxyendonuclease from calf thymus was purified to apparent homogeneity. The redoxyendonuclease recognized and induced cleavage of DNA damaged by ultraviolet light. The enzyme preparation produced a single band of a relative molecular mass of approximately 34 kDa upon SDS/PAGE. The apurinic/apyrimidinic endonuclease and the DNA glycosylase activities remained associated in the apparently homogeneous preparation of the enzyme. The redoxyendonuclease activity displayed a broad pH optimum between pH 5.0-8.5 and exhibited no requirement for divalent cations. By application of FPLC columns Mono-S, Mono-Q and Mono-P, the isoelectric point (pI) of the enzyme was found to be approximately 8.0. Using the DNA sequencing procedure of Maxam and Gilbert [Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560] the purified enzyme was found to incise ultraviolet-light-irradiated DNA at pyrimidine sites as observed previously with a more crude form of the enzyme. While the most frequently cleavaged sites for the crude preparation were at cytosine residues, the apparently homogeneous enzyme preparation frequently induced cleavage sites at both cytosine and guanine residues. Predominant incision induced by the apparently homogeneous preparation was observed at guanine residues when a particular DNA sequence was used as substrate. Furthermore, the 16 N-terminal amino acid residues of the purified enzyme were identified. The sequence did not show any significant similarity to other known proteins.     

Purification and characterization of poly(ADP-ribose) synthetase from calf thymus.

Poly(ADP-ribose) synthetase from calf thymus has been purified to apparent homogeneity by a simple and rapid method with a recovery of 10 to 20%. The enzyme activity absolutely requires the presence of DNA. Histone further stimulates the reaction. The Km for NAD and the maximal velocity at 25 degrees C and pH 8.0 in the presence of both compounds are 55 micron and 1,400 nmol/min/mg, respectively. The sedimentation coefficient (s020,w) of the enzyme is 5.80 S. The molecular weight is calculated to be 108,000 by sedimentation equilibrium method using a partial specific volume of 0.736 ml/g. This value is in good agreement with the molecular weight values of 115,000 and 120,000 determined by gel filtration on Sephadex G-200 and gel electrophoresis in the presence of sodium dodecyl sulfate, respectively. The enzyme is colorless and its absorption spectrum shows a maximum at 280 nm. From a CD spectrum, alpha helical content is estimated to be approximately 30%. The enzyme is a basic protein having a pI value of 9.8 and is rich in lysine rather than arginine. Neutral sugar, phospholipid, and DNA are not detected in the final preparation. These data indicate that the purified enzyme is a simple globular protein composed of a single polypeptide having an approximate molecular weight of 110,000.     

Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus.

We have devised a general protein fractionation procedure which selects for eukaryotic DNA-binding proteins, some of which resemble DNA-unwinding proteins from prokaryotes. Proteins were selected which (a) pass through a native DNA-cellulose column, (b) bind to a denatured DNA-cellulose column, and (c) remain bound to the latter column during a rinse with a dilute solution of the sodium salt of the polyanion dextran sulfate. When this fractionation was applied to the soluble proteins fo calf thymus, three major protein species were recovered. The predominant one has an apparent molecular weight of about 24,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is isoelectric near neutrality, and elutes as a monomer from denatured DNA-cellulose at moderate NaCl concentrations. This protein, designated calf-unwinding protein 1 (UP1), has been purified to homogeneity. However, isoelectric focusing reveals four or five subspecies (apparently separated by single-charge differences) which differ appreciably in their affinities for DNA. Two other major proteins are obtained which have apparent molecular weights in sodium dodecyl sulfate of 33,000: the first, which elutes with low salt from DNA-cellulose as a homogeneous preparation, appears to be a basic protein (although it is clearly not a histone); the other, which elutes from DNA-cellulose as the major component of a "high salt eluting fraction," is an acidic protein which co-purifies with less prominent species of higher molecular weights. Proteins similar to each of these three major calf thymus proteins have been observed by us and others in tissue culture cells of mouse, hamster, monkey, and humans, suggesting their wide occurrence among eukaryotes.     

Purification of mRNA guanylyltransferase from calf thymus.

mRNA guanylyltransferase has been extensively purified from calf thymus. A GTP-binding assay was used based on the observations by Shuman and Hurwitz (1981) and Venkatesan and Moss (1982) that vaccinia virus and HeLa cell mRNA guanylyltransferases bind the GMP moiety from GTP in the absence of an acceptor RNA. The mol. wt. of the purified enzyme from calf thymus, estimated by polyacrylamide gel electrophoresis in the presence of SDS, is 65 000. The major protein in the purified enzyme fraction comigrates with the peptide labelled with GMP. Based on scans of silver-stained polyacrylamide gels, mRNA guanylyltransferase constitutes greater than 50% of the protein in these fractions. The enzyme catalyzed the guanylylation at the 5' end of poly(A) with a mixture of diphosphate and triphosphate ends. No evidence was obtained for a direct interaction between mRNA guanylyltransferase and RNA polymerase B (II).     

Purification and characterization of an endonuclease from calf thymus acting on irradiated DNA.

An endonuclease acting on DNA exposed to ultraviolet light or gamma-rays has been extensively purified from calf thymus. The enzyme has a pH optimum at pH 7.0-7.5, acts with equal efficiency in the presence of EDTA or divalent cations (Mg-2+ or Ca-2+), is inhibited by NaCl and tRNA and is inactivated by incubation at 50 degrees C. Its molecular weight, determined by Sephadex chromatography or sodium dodecylsulfate gel electrophoresis, is approx. 30 000. The enzyme catalyzes the formation of breaks with 5'-phosphate termini in double-stranded DNA irradiated with ultraviolet or gamma-rays. It does not act on unirradiated DNA or denatured DNA. Since in all these properties the enzymatic activity on ultraviolet- and gamma-irradiated DNA behaved similarly and since the two activities cochromatographed in all systems used during purification, we conclude that they are associated with the same protein. The site of action of the enzyme in ultraviolet-irradiated DNA is a photoproduct other than pyrimidine dimers. Such a photoproduct can also be induced by irradiation of the DNA in vivo, i.e. within the cells.     

Purification and partial characterization of a DNA polymerase alpha species from calf thymus.

We have purified a DNA polymerase alpha species from calf thymus to near homogeneity. The enzyme sediments at 5.7 S and contains two polypeptides of 123000 and 134000 daltons in about equimolar ratio. The enzyme is inhibited by aphidicolin and N-ethylmaleimide, and retains its activity in buffers containing moderate salt conditions. Activated DNA is a better substrate than poly-(dA) . (dT) 10.     

DNA helicase from calf thymus. Purification to apparent homogeneity and biochemical characterization of the enzyme

We have purified a DNA helicase from calf thymus to apparent homogeneity by monitoring the activity with a strand displacement assay. DNA helicase followed the DNA polymerase alpha-primase complex through chromatography on phosphocellulose and hydroxylapatite. Separation from DNA polymerase alpha-primase complex as well as from the bulk of another DNA-dependent ATPase was achieved on heparin-Sepharose. Further purification steps included ATP-agarose and fast protein liquid chromatography-Mono S. A 47-kDa polypeptide cosedimented with the DNA helicase activity in a glycerol gradient as well as in gel filtration on Superose 6. The calf thymus DNA helicase had a sedimentation coefficient of 4-7 S and Stokes radius of about 45 A suggesting that the enzyme might be monomer in its functional form. DNA helicase activity requires a divalent cation with Mg2+ being more efficient than Mn2+ or Ca2+. Hydrolysis of ATP is required since the two nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenylyl (beta, gamma-methylene)diphosphonate cannot substitute for ATP or dATP in the displacement reaction. Calf thymus DNA helicase is able to use ATP, dATP, dideoxy-ATP, CTP, and dCTP with Km for ATP and dATP of 0.2 and 0.25 mM, respectively. The enzyme can displace a fragment of 24 bases completely in an enzyme concentration- and time-dependent manner. The DNA helicase appears to bind to single-stranded DNA and to move to single-strand double-strand transition. The directionality of unwinding is 3'----5' with respect to the single-stranded DNA to which the enzyme is bound.     

Purification and characterization of a type II DNA topoisomerase from bovine calf thymus

We report here the large scale purification of DNA topoisomerase II from calf thymus glands, using the unknotting of naturally knotted P4 phage DNA as an assay for enzymatic activity. Topoisomerase II was purified more than 1300-fold as compared to the whole cell homogenate, with 22% yield. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 125 and 140 kDa. Tryptic maps of the two bands indicated that they derive from the same protein. Using these fragments, specific polyclonal antisera to topoisomerase II were raised in rabbits. Immunoblotting of whole cell lysates from various species indicated that topoisomerase II is well conserved among mammals and has a native subunit molecular mass of 180 kDa. Analytical sedimentation and gel filtration were used to determine a sedimentation coefficient of 9.8 S and a Stokes radius of 68 A. The calculated solution molecular mass of 277 kDa implies a dimer structure in solution. The purified topoisomerase II unknots P4 DNA in an ATP-dependent manner and is highly stimulated in its relaxation activity by ATP. A DNA-stimulated ATPase activity, as has been found with other type II topoisomerases, is associated with the purified enzyme. Approximate kinetic parameters for the ATPase reaction were determined to be: a Vmax of 0.06 nmol of ATP/(micrograms of protein) (min) and Km of 0.2 mM in the absence of DNA, and a Vmax of 0.2 nmol of ATP/(micrograms of protein) (min) and Km of 0.4 mM ATP in the presence of supercoiled plasmid DNA.     

Purification and characterization of a type I DNA topoisomerase from calf thymus mitochondria

A type I topoisomerase has been purified more than 4000-fold from calf thymus mitochondria. The enzyme is membrane associated and is effectively solubilized by 1% Triton X-100 treatment of purified mitochondrial inner membranes. This ATP-independent enzyme relaxes positively and negatively supercoiled DNA with delta LK = 1. At low ionic strength, the native enzyme appears to be a monomer (sedimentation coefficient of 4.3 S and Stokes radius of 34 A), but it can form a weakly associated dimer at higher salt concentrations (sedimentation coefficient of 7.0 S and Stokes radius of 47.5 A). The mitochondrial type I topoisomerase is distinguishable from the nuclear enzyme by its (1) pH profile, (2) thermal stability, (3) response to dimethyl sulfoxide and Berenil, and (4) molecular weight. The mitochondrial enzyme is inhibited by elevated concentrations of the bacterial DNA gyrase inhibitor novobiocin, but not nalidixic or oxolinic acids. Sensitivity to N-ethylmaleimide indicates the importance of cysteine for catalytic activity. It is estimated that there are at least five copies of topoisomerase I per mammalian mitochondrion or a minimum of one to two per mitochondrial genome. In a manner similar to that observed with leukemia (nuclear and mitochondrial), calf thymus (nuclear), and HeLa (nuclear) cell type I topoisomerase, the calf thymus mitochondrial enzyme is inhibited by physiological concentrations of ATP.     

Purification of terminal riboadenylate transferase from calf thymus gland.

A poly(A) polymerase has been purified from the soluble protein fraction of calf thymus gland. The activity is cytoplasmic and nonparticulate. Mn-2+ATP is the preferred substrate. On the basis of disc gel electrophoresis in sodium dodecyl sulfate-acrylamide gels, gel filtration, and sedimentation velocity in sucrose gradients, the enzyme has a molecular weight of 62,000 and appears to consist of one polypeptide chain. The enzyme preparation is shown to be nearly homogeneous by disc gel electrophoresis and isoelectric-focusing. The activity has a pI of about 7.4. The specific activity of the enzyme is about 1700 mumol per hour per mg of protein, giving a turnover number of about 1800 mol of substrate per mol of enzyme min- minus 1. The activity is highly specific for ATP and is inhibited by other ribonucleoside triphosphates. It is sensitive to high levels of RNA-polymerase inhibitors. Km for oligoadenylate is 50 muM in the presence of Mn-2+ and 200 muM in Mg-2+ and equivalent Vmax is achieved with either metal ion. The initiator function may be filled by a variety of oligoribonucleotides having a free 3'-OH.     

Purification and characterization of a calf thymus protein active on lipid metabolism

1. This work concerns the purification of a calf thymus protein that increases the binding of human 125I-labeled low density lipoprotein (LDL) on both human skin fibroblasts and a special line of rat liver cells, BRL 3A. 2. It was found that the thymus gland affects cholesterol metabolism via an activation of the LDL receptor pathway. 3. Moreover, the thymus protein active on the LDL receptor pathway has a different amino acid composition and molecular weight from other well-characterized thymic peptides.