Calf Thymus Enzyme Research Articles

Effect of novel benzoylphenylurea derivatives on DNA polymerase alpha activity using the synthesome-based in vitro model system.

Six benzoylphenylurea (BPU) derivatives have been synthesized in Japan and extensively evaluated by the U.S. National Cancer Institute. They demonstrated potent antitumor activity in vitro against several cancer cell lines as well as in vivo against several tumor models. One of these agents, NSC639829, has now entered clinical trials. Studies have shown that these compounds are effective inhibitors of in vitro tubulin polymerization. The parent compound, NSC624548 (HO-221), has been shown to inhibit calf thymus DNA polymerase alpha activity. In this study we examined the effects of four BPU derivatives (NSC624548, NSC639828, NSC639829, and NSC654259) on the activity of the synthesome-associated DNA polymerase alpha, Escherichia coli DNA polymerase I, and calf thymus DNA polymerase alpha. Among the compounds tested, only NSC624548 and NSC639828 inhibited the activities of E. coli DNA polymerase I and calf thymus DNA polymerase alpha. Excess DNA polymerase I or DNA polymerase alpha dramatically reduced the inhibition produced by these compounds. NSC624548 and NSC639828 also showed inhibitory effects of the synthesome-associated DNA polymerase alpha similar to that produced upon using the purified E. coli and calf thymus enzymes. All of the four compounds did not show inhibitory effect on DNA polymerase delta. The similar pattern of inhibition these compounds exert on both the purified calf thymus and the synthesome-associated DNA polymerase alpha offers further support for the validity of the DNA synthesome as a novel in vitro model system for studying anticancer drug action.

Reconstitution and characterization of the human DNA polymerase delta four-subunit holoenzyme.

Mammalian DNA polymerase delta was originally characterized as a tightly associated heterodimer consisting of the catalytic subunit, p125, and the p50 subunit. Recently, two additional subunits, the third (p68) and fourth subunits (p12), have been identified. The heterotetrameric human pol delta complex was reconstituted by overexpression of the four subunits in Sf9 cells, followed by purification to near-homogeneity using FPLC chromatography. The properties of the four-subunit enzyme were shown to be functionally indistinguishable from those of pol delta isolated from calf thymus. The physicochemical properties of both the reconstituted heterotetramer and the heterodimer of the p125 and p50 subunits were examined by gel filtration and glycerol gradient ultracentrifugation. These studies show quite clearly that the heterodimer and heterotetramer complexes do not behave in solution as dimeric structures. This issue is of significance because several studies of the yeast pol delta complexes have indicated that the third subunit is able to bring about the dimerization of the pol delta complex. The heterodimer is only weakly stimulated by PCNA, whereas the heterotetramer is strongly stimulated to a level with a specific activity comparable to that of the calf thymus enzyme. These results resolve earlier, conflicting reports on the response of the heterodimer to PCNA. Nevertheless, the heterodimer does have some ability to interact functionally with PCNA, consistent with evidence that the p125 subunit itself has an ability to interact with PCNA. The functional interaction of PCNA with the pol delta complex may likely involve multiple contacts.

Effects of camptothecin analogues on DNA transformations mediated by calf thymus and human DNA topoisomerases I.

Camptothecin (CPT) and 10 structural analogues were studied to characterize their effects on specific rearrangements of DNA structure mediated by human and calf thymus DNA topoisomerases I. A 30 base pair DNA duplex containing a single high-efficiency topoisomerase cleavage site was incubated with each of the enzymes in the presence of the inhibitors. Individual inhibitors stabilized the covalent enzyme-DNA binary complex to different extents, as anticipated. However, for several of the inhibitors, the extent of ternary complex formation differed substantially for the human and calf thymus enzymes. In common with calf thymus topoisomerase I, the human enzyme was shown to mediate the rearrangement of branched, nicked, and gapped DNA substrates that constitute models for illegitimate recombination. However, some of these rearrangements proceeded with different rates and efficiencies in the presence of human topoisomerase I. When inhibition of three of the rearrangements by CPT analogues was studied, most of the analogues exhibited differential effects on a given transformation, depending on the source of the enzyme employed.

Glycosylation of RNA polymerase II from wheat germ

RNA polymerase II from wheat germ was analyzed for the presence of sugars. The two largest subunits and the 27 and 25 kDa subunits were found to be glycosylated by a variety of sugars. However, no N-acetylglucosamine was detected, which was found by Kelly et al. (J. Biol. Chem. (1993) 268, 10416–10424) in the largest subunit of RNA polymerase II from calf thymus. Thus it appears that the regulatory function of this sugar, postulated by Kelly et al., is performed in the wheat germ enzyme by other monosaccharides. Carbohydrate analysis of the two largest subunits of the calf thymus enzyme also revealed the presence, beside N-acetylglucosamine, of other sugars. Some similarities in the features of glycosylation of the two polymerases, isolated from very different organisms, suggest that the sugar moieties have an important role in the structure and/or function of these enzymes.

Stimulation of site-specific topoisomerase II-mediated DNA cleavage by an N-methylpyrrolecarboxamide-anilinoacridine conjugate: relation to DNA binding.

The DNA binding properties and effects on topoisomerase II of MePyGA, an anilinoacridine derivative bearing an N-methylpyrrolecarboxamide unit at position 1', have been compared with those of its precursor glycylanilinoacridine and the structurally related antileukaemic drug amsacrine. Electric linear dichroism spectroscopy reveals that MePyGA intercalates its acridine chromophore between DNA base pairs with a preference for GC-rich sequences, whereas both its structural analogue lacking the N-methylpyrrole unit and amsacrine intercalate into DNA without any strong sequence preference. The effects of the test drug on the catalytic activities of topoisomerase II were studied in vitro using purified calf thymus enzyme and 32P-labeled DNA. MePyGA stabilizes the topoisomerase II-DNA covalent complex and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. The removal of the N-methylpyrrole unit abolishes both the GC-preferential binding to DNA and the topoisomerase II-mediated DNA cleavage. MePyGA and amsacrine stimulate the cleavage of DNA by topoisomerase II at different places: cleavage stimulated by amsacrine is consistent with the expected adenine requirement at position +1 whereas the predominant sites of DNA cleavage stimulated by MePyGA contain a cytosine at position +/- 1. This is the first instance where an anilinoacridine derivative differing only by the nature of the substituent at position 1' has been found to affect the catalytic activity of topoisomerase II differently. The spectroscopic and biochemical data lead to the conclusion that two functional domains can be identified in MePyGA: its anilino group can be regarded as a skeletal core to which are connected (i) the tricyclic acridine moiety which represents the DNA-binding domain and (ii) the N-methylpyrrole moiety which constitutes the topoisomerase II-targeted domain. The structure of the substituent at position 1' of the anilinoacridine chromophore evidently determines the location of the sites of DNA cleavage by topoisomerase II. These findings provide guidance for the synthesis and development of new topoisomerase II-targeted antitumor anilinoacridine derivatives.

Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system

We have overproduced the full-length human poly(ADP-ribose) polymerase (PARP) in Spodoptera frugiperda (S/9) cells using a baculovirus expression vector system. Approx. 20 mg of purified protein from 5 × 10 8 S/9 cells were obtained by a simple three-step purification procedure including 3-aminobenzamide affinity chromatography. The recombinant protein (rePARP), which migrates as a unique 116-kDa band on SDS-polyacrylamide gels, was identified as PARP by Western blotting using either polyclonal antibodies raised against the purified human and calf thymus enzymes. Furthermore, rePARP is a functional protein, as demonstrated by its ability to specifically bind Zn 2+ and DNA, and to recognize single-strand breaks in DNA. The purified enzyme has the same affinity for NAD + and turnover number as the human placental PARP. Thus, rePARP produced in insect cells is biologically active and suitable for functional analysis. The reproducibility of the overproduction and the simplicity of the purification protocol, as well as the yield of the produced protein, should greatly facilitate physicochemical and structural studied.

Apparent Role of Adenosine Diphosphoribosyl Transferase in the Development of Mytilus edulis and the Inhibition of Differentiation by Ligands of the Enzyme Protein

The poly(ADP-ribose) polymerase or transferase (ADPRT) activity of developing embryos of Mytilus edulis increases with the progression of larval growth. ADPRT protein was partially purified from 2-hr-old embryos and identified by gel electrophoresis and immunotransblot, demonstrating cross-reactivity with anti-ADPRT IgG produced against the calf thymus enzyme. Two inhibitors of ADPRT, benzamide, competing with NAD at the nicotinamide binding site, and 6-amino-1,2-benzopyrone, which competes with DNA at the DNA binding site(s), both selectively arrest differentiation at the prodissoconch stage. The DNA site-oriented inhibitor, 6-amino-1,2-benzopyrone, has a much larger differentiation arresting effect than benzamide. The arrest of differentiation by 6-amino-1,2-benzopyrone is reversible. A probable ecotoxicity of ADPRT ligands on mussel differentiation is proposed.

Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon.

DNA polymerase epsilon, formerly known as a proliferating cell nuclear antigen-independent form of DNA polymerase delta, has been shown elsewhere to be catalytically and structurally distinct from DNA polymerase delta. The catalytic activity of HeLa DNA polymerase epsilon, an enzyme consisting of greater than 200- and 55-kDa polypeptides, was assigned to the larger polypeptide by polymerase trap reaction. This catalytic polypeptide was cleaved by incubation with trypsin into two polypeptide fragments with molecular masses of 122 and 136 kDa, the former of which was relatively resistant to further proteolysis and possessed the polymerase activity. The cleavage increased the polymerase and exonuclease activities of the enzyme some 2-3-fold. DNA polymerase epsilon was also purified in a smaller 140-kDa form from calf thymus. The digestion of this form of the enzyme by trypsin also generated a 122-kDa polypeptide. These results suggest that the catalytic core of DNA polymerase epsilon is a 258-kDa polypeptide that is composed of two segments linked with a protease-sensitive area. One of the segments harbors both DNA polymerase and 3'----5' exonuclease activities. In spite of the different polypeptide structures, the catalytic properties of the HeLa enzyme, its trypsin-digested form, and the calf thymus enzyme remained essentially the same.

Isolation of cDNA clones encoding an enzyme from bovine cells that repairs oxidative DNA damagein vitro: homology with bacterial repair enzymes

Ionizing radiation and radiomimetic compounds, such as hydrogen peroxide and bleomycin, generate DNA strand breaks with fragmented deoxyribose 3' termini via the formation of oxygen-derived free radicals. These fragmented sugars require removal by enzymes with 3' phosphodiesterase activity before DNA synthesis can proceed. An enzyme that reactivates bleomycin-damaged DNA to a substrate for Klenow polymerase has been purified from calf thymus. The enzyme, which has a Mr of 38,000 on SDS-PAGE, also reactivates hydrogen peroxide-damaged DNA and has an associated apurinic/apyrimidinic (AP) endonuclease activity. The N-terminal amino acid sequence of the purified protein matches that reported previously for a calf thymus enzyme purified on the basis of AP endonuclease activity. Degenerate oligonucleotide primers based on this sequence were used in the polymerase chain reaction to generate from a bovine cDNA library a fragment specific for the 5' end of the coding sequence. Using this cDNA fragment as a probe, several clones containing 1.35 kb cDNA inserts were isolated and the complete nucleotide sequence of one of these determined. This revealed an 0.95 kb open reading frame which would encode a polypeptide of Mr 35,500 and with a N-terminal sequence matching that determined experimentally. The predicted amino acid sequence shows strong homology with the sequences of two bacterial enzymes that repair oxidative DNA damage, ExoA protein of S. pneumoniae and exonuclease III of E. coli.

Two forms of DNA polymerase delta from mouse cells. Purification and properties.

A procedure is described for the purification from cultured mouse cells of two DNA polymerase "delta-like" enzymes, as defined by intrinsic 3'-exonuclease activity, inhibition by aphidicolin, and relative insensitivity to N2-(p-n-butylphenyl)-dGTP. One of the two enzymes has been purified to near homogeneity and, similar to the DNA polymerase delta from calf thymus described by Lee et al. (Lee, M. Y. W. T., Tan, C. K., Downey, K. M., and So, A. G. (1984) Biochemistry 23, 1906-1913), it has a total molecular mass of 178 kDa (from sedimentation velocity of 8.0 S and Stokes radius of 54 A) and is composed of one each of 125- and 50-kDa polypeptides. It also resembles the DNA polymerase delta of Lee et al. in being stimulated by proliferating cell nuclear antigen (PCNA). It is the first clear structural and functional counterpart of the calf thymus enzyme. The major difference between the mouse DNA polymerase delta and the calf thymus enzyme of Lee et al. is that, under specific conditions, the mouse enzyme is active with poly(dA).oligo(dT) in the absence of PCNA, whereas the activity of the calf thymus enzyme with this template is reported to be completely dependent on PCNA. The reason for this difference is not known at this time. The second mouse cell enzyme has a molecular mass of 112 kDa (from sedimentation velocity of 6.3 S and Stokes radius of 43.0 A) and consists of a single polypeptide of 123-125 kDa in denaturing gels (p125). On the basis of its apparent formation by dissociation of DNA polymerase delta, and multiple similarities with DNA polymerase delta in enzymatic properties, the p125 is provisionally identified as the 125-kDa polypeptide of DNA polymerase delta. The p125 does not respond to PCNA, suggesting that the 50-kDa polypeptide is required for the stimulation of DNA polymerase delta by PCNA. The presence of the p125 in cell extracts would explain reports that DNA polymerase delta consists of a single polypeptide of approximately 125 kDa and/or thast it has a smaller molecular mass than DNA polymerase delta of Lee et al. and is not affected by PCNA (this does not apply to PCNA-independent DNA polymerase delta-like enzymes with higher molecular mass than the polymerase delta of Lee et al., which have recently been named DNA polymerases epsilon).

Resolution of synthetic Holliday junctions in DNA by an endonuclease activity from calf thymus.

Extracts of calf thymus have been fractionated to reveal a nuclease activity that specifically cleaves model Holliday junctions in vitro. The products of cleavage are unbranched linear duplex DNA molecules. Using synthetic four-way junctions, we show that the major sites of cutting are diametrically opposed, at sites one nucleotide from the base of the junction. Other types of four-way junctions, including pseudo-cruciform structures and cruciforms extruded from supercoiled plasmids, are also cleaved by the nuclease. The Mr of the partially purified activity, determined by gel filtration, is approximately 75,000. The calf thymus enzyme provides the first example of an endonuclease from a higher eukaryote that acts specifically on branch points in DNA, and indicates that junction-resolving proteins are normal constituents of somatic cells.

Suppression of nuclear ADP-ribosyltransferase activity in Ehrlich ascites tumor cells by 5-azacytidine: Modification of DNA as a cause of suppression

Exposure of Ehrlich ascites tumor cells to 5-azacytidine for 5 h resulted in a partial loss of ability of DNA to stimulate ADP-ribosyltransferase activity, as assessed in a reconstituted in vitro enzyme system consisting of purified calf thymus enzyme, calf thymus whole histone and DNA isolated from the cells. The degree of suppression in vitro varied depending on the amount of histone and DNA added and it reached a maximum with a value of 83% and 62% of control for DNAs from cells exposed to 10 μM and 30 μM 5-azacytidine, respectively, at a histone/DNA mass ratio of 0.4. In the absence of histone (conditions of auto-ADP-ribosylation of the enzyme), no suppression was detectable.

DNA polymerase α-DNA primase from human lymphoblasts

The DNA polymerase α-DNA primase complex from the human lymphoblast line HSC93 has been enriched to near homogeneity by using an immunoaffinity purification protocol which was developed earlier for the purification of the calf thymus enzyme (Nasheuer, H.-P. and Grosse, F. (1987) Biochemistry 26, 8458–8466). Immunoaffinity purified polymerase-primase from human cells consisted of four subunits displaying molecular weights of 195000 and 180000 for the DNA synthesizing α-subunit, of 68000 for the β-subunit, and of 55000 and 48000 for the primase-carrying γ- and δ-subunit, respectively. The isoelectric pH values for the individual subunits were estimated from non-equilibrium pH gradients to be between 5.9 and 5.7 for the α-subunit, at 5.5 for the β-subunit, and at 7.5 and 8.0 for the γ- and δ-subunit, respectively. The purified polymerase-primase converted single-stranded ΦX174 DNA into the double-stranded form in a primase-initiated reaction. During this process, 3–10 RNA primers were formed. RNA primers were about 11 nucleotides long. Elongation of existing RNA primers by the human polymerase-primase was semi-processive; following primer binding the DNA polymerase continuously incorporated 20 to 50 nucleotides, then it dissociated from the template DNA.

Purified RNA polymerase II recognizes specific termination sites during transcription in vitro.

We have studied the ability of certain well-defined prokaryotic DNA sequences to act as specific termination signals for highly purified calf thymus RNA polymerase II. We used duplex DNA fragments modified to direct efficient and specific transcription of defined DNA templates to follow transcription with RNA polymerase alone in the absence of additional protein factors. Elongation of RNA chains by RNA polymerase II is processive through most DNA sequences. However, certain DNA sequences serve as effective "intrinsic" terminators for RNA polymerase II; in this they resemble the "rho-independent" terminators for the bacterial RNA polymerase. Several rho-independent bacterial terminators are also able to act as termination signals for RNA polymerase II. However, there is no apparent correlation between the efficiency of termination for the bacterial enzyme and that found for the calf thymus enzyme. One very efficient bacterial terminator (phage T7 early terminator) gives no termination with RNA polymerase II, and we have identified at least two sites that cause the eukaryotic enzyme to terminate but have no effect on transcription by the bacterial enzyme. Hence, the signals recognized as intrinsic termination sites for the two enzymes are substantially different. All of the sites that act as intrinsic terminators for RNA polymerase II contain a series of consecutive thymidine residues in the nontranscribed DNA strand (T-run), and the 3' end of the completed RNA normally lies within this sequence. It is plausible that the T-run is part of the signal for an RNA polymerase II termination site; however, there is no apparent correlation between the number of T residues and the efficiency of the terminator, suggesting that other sequence elements are required for, or modulate, termination. Several lines of evidence suggest that the formation of RNA secondary structures in the nascent transcript is not an essential element of the intrinsic RNA polymerase II termination signal.

Mechanism of action of a mammalian DNA repair endonuclease

The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined. The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents. The calf thymus enzyme incises damaged DNA at sites of pyrimidines. A cytosine photoproduct was found to be the primary monobasic UV adduct. The calf thymus endonuclease and E. coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms. The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage. The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity. The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III. The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups. These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes. We propose the name redoxy endonucleases for this group of enzymes.

Casein kinase type II is involved in the inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole of specific RNA polymerase II transcription.

We have described a HeLa protein kinase whose activity is inhibited by the nucleotide analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) at concentrations similar to those required to inhibit in vivo and in vitro specific transcription (Zandomeni, R., and Weinmann, R. (1984) J. Biol. Chem. 259, 14804-14822). We have now detected an analogous DRB-sensitive kinase from calf thymus and purified it to homogeneity. Based on the subunit composition of the enzyme and other common biochemical and chromatographic properties, we identified it as casein kinase II. The extent of DRB inhibition of the purified calf thymus enzyme is indistinguishable from that observed for inhibition of in vitro transcription with the HeLa cell extract. The DRB bromo- derivative, 5,6-dibromo-1-beta-D-ribofuranosylbenzimidazole is a more potent inhibitor of in vivo transcription and inhibits purified casein kinase II activity and specific in vitro transcription at 6-10 times lower concentrations than DRB. Moreover, addition of an excess of the purified calf thymus casein kinase II enzyme to a HeLa in vitro transcription reaction inhibited by DRB partially overcomes this inhibition. Thus, we conclude that casein kinase II is involved directly or indirectly in the inhibition by DRB of specific RNA polymerase II-mediated transcription. This demonstrates the participation of a protein kinase in a eukaryotic RNA polymerase II-specific transcription system.

Expression of human terminal deoxynucleotidyl transferase in Escherichia coli.

A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA. A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106. DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides. pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein. pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711. Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked. N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence. The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106. Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined. The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene. Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity. Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.

Monoclonal antibodies to human terminal transferase.

Human terminal deoxynucleotidyltransferase was used to produce mouse monoclonal antibodies. Three different assays were used to screen for terminal transferase antibodies and antibody-producing hybridoma cells: an enzyme binding assay, an enzyme-linked immunosorbent assay, and a "terminal transferase-specific antibody" cytochemical procedure that allows visualization of the homogeneity of the hybridoma clones producing antibodies to terminal transferase. Of the 12 mouse hybridoma clones isolated, seven produce IgG1 and five produce IgG2a immunoglobulins. Only one of these monoclonal antibodies is inhibitory to human terminal transferase activity. Four of the monoclonal antibodies react with terminal transferase peptides after separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the remainder appear to recognize only native determinants on the human enzyme. Cross-reactivity studies with purified calf thymus enzyme show that although four of the monoclonal antibodies bind to native enzyme, none react with calf thymus terminal transferase peptides after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting structural differences within the cross-reactive determinants. A more general survey on the affinity of these monoclonal antibodies to human terminal transferase for the enzyme from other species using an enzyme-linked immunosorbent assay demonstrates that none of the monoclonal antibodies reacts with chicken thymus terminal transferase. The affinity of binding for terminal transferase from other mammals to this panel of monoclonal antibodies varies with the clones as expected, but few bind as strongly as the human enzyme. These results suggest that although antigenic determinants are conserved through evolution, fine structural changes within the determinants do occur.

DNA ligase from mouse Ehrlich ascites tumor cells.

The molecular (Mr = 120,000; s20, w = 5S) and catalytic properties (Km (ATP) = 3 microM; Km (nicked DNA) = 0.2 microM; Km (Mg2+) = 3 mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.

Specific termination of in vitro transcription by calf thymus RNA polymerase III.

In vitro transcription of cleaved SV40 DNA with calf thymus RNA polymerase reveals a discrete transcript. The pattern of resistance to the inhibitor alpha-amanitin identifies the RNA as a product of RNA polymerase III transcription. The RNA is shown to initiate artificially near a DNA terminus created by cleavage and to terminate specifically near a cluster of 8 thymidine residues within the SV40 control region. Faithfully initiated transcripts cannot be detected using the calf thymus enzyme, supporting the idea that polymerase III termination can be accomplished by an initiation-deficient enzyme. Transcription of SV40 DNA in a HeLa cell lysate also leads to specific polymerase III transcription. When PvuII-cleaved DNA is the template, the same RNA is produced as with the calf thymus enzyme. At the lowered lysate concentration known to activate certain AluI-family transcripts, a collection of SV40 polymerase III transcripts is also produced. These do not depend on restriction cleavage of the DNA and thus arise from transcription of intact DNA.