Superhelical DNA-dependent ATPase from calf thymus

Two physically and catalytically distinct DNA-dependent ATPases were isolated from a purified preparation of calf thymus poly(ADP-ribose) polymerase. A unique feature of these two ATPases was the high stimulation by supercoiled DNA. Other nucleic acids (including denatured DNA and ribosomal RNA) and certain polynucleotides differentially stimulated the two enzymes. We have not detected any other DNA-related activity associated with these ATPases.     

Histone III. VI. Two forms of calf thymus histone III.

Microheterogeneity in the amino acid sequence of pea embryo histone III at residue 96 has been established previously. It has been indicated that calf-thymus contains two forms of histone III, with 1 or 2 residues of cysteine, respectively. Evidence is presented here that these two forms are also due to microheterogeneity at residue 96 with one form containing a cysteine residue and the other a serine residue.     

N-terminal acetyl-peptides from two calf thymus histones

1. The same N-terminal peptide was isolated from two major calf thymus histone fractions, F2al and F2a2. 2. The structure of the peptides is N-acetylseryl-glycyl-arginine, and the yields account for most of the N-acetyl groups previously found in these histones. 3. Some other peptides not giving a ninhydrin reaction were also investigated.     

Characterization of DNA kinase from calf thymus

DNA kinase has been purified to homogeneity from calf thymus. The purified enzyme, with a specific activity of 16.7 units/mg protein at 25 degrees C, exhibited a sharp pH/activity curve with a pH optimum at 5.5 and low activity at alkaline pH. The molecular weight of the enzyme was estimated by dodecylsulfate/polyacrylamide gel electrophoresis to be 5.4 X 10(4). The enzyme has a sedimentation coefficient of 4.0 S. An apparent molecular weight of 5.6 X 10(4) and a Stokes' radius of 3.3 nm were estimated by gel-filtration on Sephadex G-100. The enzyme phosphorylates neither yeast RNA nor poly(A) instead of DNA. Compared with rat liver DNA kinase, calf thymus DNA kinase is relatively resistant to the inhibition by sulfate (Ki = 7 mM) and pyrophosphate (Ki = 5 mM). The enzyme activity is markedly stimulated by polyamines at the sub-optimal concentration of Mg2+ but not by monovalent cations.     

Synthetic RNA-dependent DNA polymerase from calf thymus

A RNA dependent-DNA polymerase was purified about 450-fold from the soluble fraction of calf thymus. This enzyme was able to copy the polyribonucleic acid strand of synthetic ribonucleic acid primed with complementary oligodeoxynucleotides, i.e., poly(rA)·(dT)₁₀. This enzyme activity was separated from the DNA-dependent DNA polymerases by both DEAE-cellulose columm chromatography and glycerol gradient centrifugation. Some properties of this enzyme were described.     

Two DNA ligase activities from calf thymus

Cell extracts from calf thymus contain two DNA ligase activities, separable by hydroxyapatite chromatography and by gel filtration. Their molecular weights, as estimated from sedimentation coefficients and Stokes radii, are M = 175,000 and M = 85,000, respectively. The two activities both require Mg⁺⁺ and ATP as cofactors, and convert nicked circular DNA molecules to a covalently closed form. The larger of the two ligase activities is more heat-stable than the smaller one, and is also active over a broader pH range.     

Histone H4 from cuttlefish testis is sequentially acetylated. Comparison with acetylation of calf thymus histone H4

The differently acetylated subfractions of histone H4 isolated from cuttlefish testis and from calf thymus were separated by ion exchange chromatography on sulfopropyl-Sephadex, using a shallow linear gradient of guanidine hydrochloride in the presence of 6 M urea at pH 3.0. The tetra-, tri-, di-, mono-, and nonacetylated forms of cuttlefish H4 represent 2, 6.4, 18, 32.2, and 41.4% of the whole histone, respectively. The di-, mono-, and nonacetylated forms of calf H4 represent 11.7, 41.3, and 44% of the whole histone, respectively. The acetylation sites were determined in each subfraction by identification of the acetylated peptides. In each acetylated H4 subfraction, the acetylated tryptic peptides were identified by peptide mapping and amino acid analysis with reference to the peptide map of nonacetylated H4. In cuttlefish testis H4, lysine 12 is the main site of acetylation in the monoacetylated subfraction; lysines 5 and 12 are found acetylated in diacetylated H4; lysines 5, 12, and 16 are found acetylated in triacetylated H4. From these results and the stoichiometry of the different H4 subfractions, it can be concluded that lysine 5 is acetylated after lysine 12. In calf thymus, lysine 16 is the only site of acetylation in the monoacetylated subfraction. All the diacetylated forms are acetylated in lysine 16, the second site of acetylation being, in decreasing order, lysine 12, lysine 5, or lysine 8. These observations suggest that acetylation occurs in a sequential manner. Moreover, the sites of acetylation depend upon the biological event in which acetylation is involved.     

Four different DNA helicases from calf thymus.

Using a strand displacement assay we have followed DNA helicase activities during the simultaneous isolation of several enzymes from calf thymus such as DNA polymerases alpha, delta, and epsilon, proliferating cell nuclear antigen, and replication factor A. Thus we were able to discriminate and isolate four different DNA helicases called A, B, C, and D. DNA helicase A is identical with the enzyme described earlier (Th?mmes, P., and Hübscher, U. (1990) J. Biol. Chem. 265, 14347-14354). The four enzymes can be distinguished by (i) their putative molecular weights after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) glycerol gradient sedimentation under low and high salt conditions, (iii) sensitivity to salt, (iv) binding to DNA, (v) nucleoside- and deoxynucleoside 5'-triphosphate requirements, and (vi) by their direction of movement. DNA helicase A unwinds in the 3'----5' direction on the DNA it was bound to, while DNA helicases B, C, and D do so in the 5'----3' direction. DNA helicase D, and to some extent DNA helicases B and C, are able to unwind long substrates of more than 400 nucleotides. Replication factor A, a single-stranded heterotrimeric DNA binding protein involved in cellular DNA replication and DNA repair stimulates the DNA helicases. The stimulatory effect is most pronounced on DNA helicase A, where replication factor A enables this helicase to unwind longer substrates. DNA helicases B, C, and D are also stimulated by replication factor A. The effect of replication factor A appears to be specific since corresponding single-stranded DNA binding proteins from Escherichia coli and bacteriophage T4 have no or even a negative effect on the four DNA helicases. Heterologous human replication factor A has no stimulatory effect on any of the four DNA helicases suggesting a species specificity of these interactions. Thus it appears that mammalian cells possess, as does E. coli, a variety of different enzymes that can transiently abolish the double helical DNA structure in the cell.     

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).