Characterization of catalytic carboxylate triad in non-replicative DNA polymerase III (pol E) of Geobacillus kaustophilus HTA.

Three aspartic acid residues D378, D380 and D531 form the catalytic carboxylate triad in Geobacillus kaustophilus (Gka) DNA polymerase III α-subunit homolog, pol E. We cloned, expressed and purified wild type (WT), alanine (D → A) and glutamate (D → E) mutant enzymes of D378, D380 and D531. The WT and mutant enzymes were biochemically characterized for DNA binding, dNTP binding and catalytic activity in the presence of two metal ions (Mg2+ and Mn2+). The polymerase activity of all mutant enzymes was lost in the presence Mg2+, whereas D378E and D531E mutant enzymes showed about 35 and 60 percent activity, with Mn2+. D380E mutant enzyme did not show noticeable activity with either metal ions suggesting its absolute requirement in polymerase reaction. Kinetic characterization of individual mutant proteins showed that the template-primer binding affinity (KD.DNA) did not change due to both D → A or D → E mutation. The KM.dNTP for D378E and D531E increased by about 10- and 100-fold, compared to WT enzyme implicating the function of these residues in dNTP binding. Based on these results and the analysis of the available crystal structures of the homologous enzyme species in their apo and E.DNA.dNTP ternary complex forms, we conclude that D378 and D531 are mainly responsible for the binding of metal chelated substrate dNTP, while D380 is solely responsible for the chemical step of phosphodiester bond formation.

A unified DNA- and dNTP-binding mode for DNA polymerases.

Crystal structures of various DNA polymerases show a common structural topology that resembles a right hand and has distinct finger, palm and thumb subdomains. Early models of the klenow fragment (KF) of Escherichia coli polymerase I showed DNA entering a large cleft that faces the palm subdomain where the catalytic site is situated1,2. However, subsequent resolution of the structures of HIV-1 reverse transcriptase, KF and polymerase beta (pol beta) bound to DNA3-5 yielded conflicting data that suggested a different orientation for DNA bound to pol beta compared with DNA bound to other polymerases. The debate, on the correct orientation of the template-primer DNA, that followed failed to reach a consensus. Using an alternative superposition scheme, we now provide convincing evidence for a common DNA-binding mode that is applicable to all polymerases, including pol beta.

Divalent cation-dependent pyridoxal 5'-phosphate inhibition of Rauscher leukemia virus DNA polymerase: characterization and mechanism of action.

We have shown that pyridoxal 5'-phosphate is an effective inhibitor of Rauscher leukemia virus DNA polymerase (Biochemistry 15 (1976) 3620). Detailed studies of this inhibition revealed that, in addition to the phosphate and aldehyde groups of pyridoxal phosphate, the presence of a divalent cation is essential for the inhibitory action. The synthesis directed by template primers containing GC base-pairs exhibited more resistance to pyridoxal phosphate inhibition than did that directed by AT base-paired templates. Maximal inhibitory activity of pyridoxal phosphate, however, is noted in the presence of Mn2+, irrespective of which template-primer is used to direct the DNA synthesis. The action of pyridoxal phosphate on the substrate binding site may be deduced from the observations that: (a) only the substrate triphosphate is able to reverse the pyridoxal phosphate-mediated inhibition; (b) the inhibition kinetics exhibit a classical competitive pattern with the substrate; (c) analogous to substrate deoxynucleoside triphosphates the inhibitor is also accepted only in the form of its divalent metal ion complex; and (d) substrate site-specific labeling of RLV DNA polymerase has been shown to occur by linking covalently the pyridoxal phosphate bound to a lysine residue at the substrate binding site.

The mechanism of inhibition of avian myeloblastosis virus reverse transcriptase by a dialdehyde derivative of ATP. Inactivation of essential sulfhydryl group function.

The dialdehyde derivative of ATP inhibits DNA synthesis by AMV reverse transcriptase, while the polymerase-associated ribonuclease H activity is significantly resistant to this reagent. Neither ATP nor its dialcohol form effectively block DNA synthesis, indicating that the aldehyde moiety is required for inhibition. The nature of the reactivity of dialdehyde-ATP with AMV reverse transcriptase has been examined and we find that: (a) inhibition is non-competitive with respect to substrate deoxynucleoside triphosphate concentration, suggesting that dialdehyde-ATP does not react at the substrate binding site; (b) pretreatment of enzyme with dialdehyde-ATP or sulfhydryl group binding reagents results in the complete loss of its template binding activity; however, treatment of preformed enzyme-template-primer complex with both inhibitors did not dissociate this complex; (c) the inhibitory effect of dialdehyde-ATP was completely reversed upon addition of reducing agents, such as dithiothreitol and sodium borohydride, indicating that dialdehyde-ATP reacts with the sulfhydryl groups present in AMV reverse transcriptase; (d) comparative studies carried out with the classical sulfhydryl reagent, dithiobisnitrobenzoic acid, revealed a remarkable similarity in its action to that of dialdehyde-ATP. We therefore conclude that the dialdehyde-ATP-mediated inhibition of AMV DNA polymerase is effected via blockage of essential sulfhydryl groups present in the enzyme protein.

Template-specific requirements for DNA synthesis by the Mason-Pfizer monkey virus DNA polymerase: unique aspects.

The biochemical properties of DNA polymerase purified from Mason-Pfizer monkey virus were studied, with respect to synthetic and natural template-primer utilization. Thes studies revealed the following new information about the Mason-Pfizer monkey virus enzyme: (a) Mason-Pfizer monkey virus polymerase was found to prefer template: primer molar nucleotide ratios of 2.5-5: 1 for optimal rates of synthesis with poly(C) .(dG)12-18 as template-primer. (b) Poly(A)-directed synthesis was stimulated by the addition of low concentrations of inorganic phosphate to the reaction mixture. (c) Poly(2' -O-methyl-cytidylate), poly(rCm), was the only template studied for which Mn2+ proved the preferred divalent cation. Combinations of divalent cations stimulated rather than inhibited poly(rCm)-directed poly(dG) synthesis by the Mason-Pfizer monkey virus enzyme. (d) Heteropolymeric regions of rabbit globin mRNA and avian myeloblastosis virus 70 S RNA could be copied by the Mason-Pfizer monkey virus polymerase with oligo(dT), oligo(U) or in the case of avian myeloblastosis virus RNA, endogenous primers. In all such studies, Mg2+ was the preferred divalent cation and a distinct preference for the DNA primer in the reverse transcription of natural RNAs was observed. These new findings necessitated comparative studies with the DNA polymerases from Rauscher murine leukemia virus and murine mammary tumor virus, as representative type C and type B retroviruses. Although the Mason-Pfizer monkey virus enzyme was found to share some properties in common with both type C and type B mammalian viral enzymes, certain of the above properties rendered it unique among the polymerases examined.

Inhibition of terminal deoxynucleotidyl transferase by adenine dinucleotides. Unique inhibitory action of Ap5A.

Terminal deoxynucleotidyltransferase (TdT) exhibits strong sensitivity to ATP and its dinucleotide analogues, Ap2A, Ap3A, Ap4A, Ap5A and Ap6A. Similar to ATP, all of the dinucleotides appear to be competitive inhibitors of TdT catalysis with respect to substrate deoxynucleoside triphosphates and effectively block the UV-mediated substrate cross-linking to TdT. Among the various dinucleotides, Ap5A and Ap6A (diadenosine 5'-5' penta- and hexaphosphate, respectively) are significantly more effective than dinucleotides containing 2, 3 or 4 phosphate backbones. Furthermore, Ap5A is found to be the only dinucleotide which has reactivity at both substrate- and primer-binding domains in TdT.

A micromethod for determination of terminal deoxynucleotidyl transferase (TdT) in the diagnostic evaluation of acute leukemias.

A micromethod for the determination of TdT in peripheral leukocytes and bone marrow cells has been developed that allows unequivocal identification and quantitation of TdT in less than 1 X 10(6) leukocytes from ALL patients, i.e., in 1 ml of peripheral blood and/or 0.5 ml of bone marrow obtained during routine clinical sampling. The method involves disruption of cell pellet with high salt and detergent followed by centrifugation of extracts at 12,000 X g and partial purification on phosphocellulose matrix by a batch elution technique using a standard laboratory microcentrifuge. Using this microassay, TdT activities have been determined in 500 samples of peripheral blood and bone marrow of 240 adult patients with acute leukemias (86 ALL, 108 ANLL, 44 blastic CML, two acute leukemias following P. vera). From an analysis of our data based on TdT activity, cell surface markers and growth patterns in soft agar and observations published in the literature, it can be concluded that the frequencies of TdT + phenotypes in the various clinical-morphological diagnostic groups are approximately 95% in ALL, 10% in ANLL, 50% in AUL, and 35% in blastic CML. Since the presence of high TdT activity is clearly associated with clinical response to specific forms of chemotherapy in blastic CML and most probably, also in ANLL, the determination of TdT should be considered in all cases of acute leukemias to objectively define prognostically important subgroups which can not be diagnosed by conventional means.

Cellular ras protooncogene expression in human mammary explant cultures. A potential marker for chemical carcinogenesis.

The major findings of the present study can be summarized as follows: 1. The newly developed assay for quantitative determination of ras protooncogene expression which utilizes affinity labeling of ras p21 with [alpha-32P]GTP can effectively demonstrate the presence of ras protooncogene expression in explant cultures of human mammary tissues. 2. The prototype chemical carcinogens NMU and BP induce amplified expression of ras protooncogene in benign (noninvolved) human mammary TDLU. 3. The specific enhancement of ras expression by BP in TDLU (target tissue) but not in MF (nontarget tissue) for tumorigenesis indicates that the constitutive levels of ras protooncogene in the target tissue are responsive to carcinogenic insult. 4. The relative extent of ras protooncogene expression may constitute a sensitive marker for target tissue susceptibility to chemical carcinogenesis.

Binding of DNA to large fragment of DNA polymerase I: identification of strong and weak electrostatic forces and their biological implications.

Examination of the electrostatic potential of a modeled complex, consisting of the Klenow fragment of E. coli DNA polymerase I and DNA template-primer, suggested the presence of two distinct interacting regions. The one displaying a strong electropositive potential field is generated by side chains of basic amino acid pairs and is directed towards the major groove site in DNA. The second electrostatic potential field around DNA is somewhat weaker and appears to be exerted by a pair of vicinal side chains of acidic and basic amino acids. The distribution of charges in this manner appears well suited for the binding of enzyme to the template-primer required in the enzymatic synthesis of DNA.

Modulation in the expression of murine mammary tumor virus, ras proto-oncogene, and of alveolar hyperplasia by fatty acids in mouse mammary explant cultures.

Hyperplastic alveolar nodule, present in the young adult non-lactating but mammary tumor virus harbouring mice (RIII), are biologically active precursor lesions which form mammary tumors at certain age. Using mammary explant cultures from these mice as a model for preneoplastic hyperplasia, we have examined the effects of various fatty acids on the changes in the cellular and molecular markers in vitro. We find that exposure of cultures to omega-6 fatty acids results in -13 fold increase on RT activity, -3 fold increase in expression of c-ras Hp21, and 2 fold increase in the frequency of lactogenic hormone-independent mammary alveolar lesions, MAL. In contrast, exposure of the cultures to eicosapentanoic or stearic acid results in no increase in RT activity, 3-fold decrease in expression of C-ras Hp21, and about 2-fold decrease in MAL frequency. These results suggest that tumor modulating effects of dietary fatty acids may be operative during the initiational and promotional events of viral carcinogenesis, well before the emergence of mammary tumors.

Biochemistry of terminal deoxynucleotidyltransferase: mechanism of inhibition by adenosine 5'-triphosphate.

The polymerization of deoxyribunucleoside triphosphate catalyzed by terminal deoxyribonucleotidyltransferase (TdT, EC 2.7.7.31) is severely inhibited by the addition of ribonucleoside triphosphates, ATP being the most potent inhibitor. Examination of the inhibitory effect of ATP using oligo(dA)12-18 as well as activated DNA as primers revealed that (a) ATP inhibition is not due to its addition onto a 3'-OH primer terminus ad judged by the lack of incorporation of labeled ATP, although under similar conditions incorporation of GTP can be demonstrated, (b) a consistent degree of inhibition was noted independent of primer or enzyme concentration; (c) addition of ATP to an ongoing reaction promptly reduces the rate of polymerization; (d) kinetic studies indicate a competitive (with respect to substrate deoxy triphosphate) pattern of inhibition; (e) addition of excess deoxyribotriphosphate promptly relieves the inhibition. Unlike ATP, other ribotriphosphates yield a mixed pattern of inhibition partly mediated by competitive mechanisms. GTP and CTP and to a minor extent UTP are incorporated into DNA in the presence or absence of deoxy triphosphate. Furthermore, addition of ATP also inhibits incorporation of GTP and CTP.

Observations on the pyridoxal 5'-phosphate inhibition of DNA polymerases.

Pyridoxal 5'-phosphate at concentrations greater than 0.5 mM inhibits polymerization of deoxynucleoside triphosphate catalyzed by a variety of DNA polymerases. The requirement for a phosphate as well as aldehyde moiety of pyridoxal phosphate for inhibition to occur is clearly shown by the fact that neither pyridoxal nor pyridoxamine phosphate are effective inhibitors. Since the addition of nonenzyme protein or increasing the amount of template primer exerted no protective effect, there appears to be specific affinity between pyridoxal phosphate and polymerase protein. The deoxynucleoside triphosphates, however, could reverse the inhibition. The binding of pyridoxal 5'-phosphate to enzyme appears to be mediated through classical Schiff base formation between the pyridoxal phosphate and the free amino group(s) present at the active site of the polymerase protein. Kinetic studies indicate that inhibition by pyridoxal phosphate is competitive with respect to substrate deoxynucleoside triphosphate(s).

Purification of nucleotide-linked peptide.

Affinity labeling of nucleotide-binding enzymes/proteins with 32P-labeled nucleotides is a powerful technique to identify nucleotide-binding proteins as well as to radiolabel the specific binding site. We have used this approach for labeling a nucleotide-binding domain in DNA polymerase and have isolated peptides bearing the linked nucleotides. The method used for separating tryptic peptides on hydrophobic matrices with an acetonitrile gradient in 0.1% trifluoroacetic acid as eluent results in loss of radioactivity, presumably through dissociation of the cross-linked nucleotide. This can be averted by the use of a non-acidic medium in the peptide purification protocol. We have devised a relatively simple procedure to concentrate the nucleotide-linked peptides by chromatography on DEAE-Sephadex A25. Most neutral and basic peptides as well as free nucleotides are removed by eluting the DEAE-Sephadex column with 0.2 M ammonium bicarbonate. The nucleotide-linked peptide is then eluted with 0.6 M ammonium bicarbonate. Radioactivity in the collected fractions is conveniently determined by scintillation counting. Labeled peptide in the 0.6 M ammonium bicarbonate eluate can be purified on a C4 reversed-phase column with an acetonitrile gradient in phosphate buffer (pH 6.8). By this procedure, 32P-labeled nucleotide linked with protein/peptide can be quantitatively purified with minimum loss.

Purification of high molecular mass species of calf thymus terminal deoxynucleotidyltransferase.

We have developed a simplified column chromatographic procedure for the simultaneous purification of two high molecular mass forms (58 kd and 45 kd) and a standard two subunit 44 kd from of terminal deoxynucleotidyltransferase (TdT) from calf thymus chromatin. The procedure involves high salt extraction of the chromatin fraction followed by successive chromatographies on phosphocellulose, DEAE sephadex, and hydroxylapatite matrices. While all 3 species of TdT comigrate throughout these steps, separation of individual species is achieved on a single stranded DNA agarose column. The combined yield of the 45 kd and 58 kd TdTs is quite high (approximately 8 mg/5000g tissue), 45 kd being the major species (approximately 60%) and the 58 kd constituting about 30%. The 44 kd species containing two subunits usually represents under 10% of the total. All the three forms of TdT showed similar specific activity and preference for purine deoxynucleoside triphosphates (dNTPs). The Km for individual dNTP with all three species of TdT is quite similar and decreases in the order dCTP greater than dTTP greater than dATP greater than dGTP. The Km for both synthetic primer and activated DNA with the 3 TdTs was, in increasing order, two subunit 44 kd less than 45 kd less than 58 kd TdT. Both 58 kd and 45 kd TdT displayed two optima for Mn++ (0.1 mM and 1 mM) and a single sharp optimum for Mg++ (2.5 mM). The two subunit 44 kd TdT exhibited a single but broad optimum for Mn++ (1 mM) and for Mg++ (10 mM).

An affinity labeling of ras p21 protein and its use in the identification of ras p21 in cellular and tissue extracts.

We have carried out photoaffinity labeling of the ras p21 protein, a ras oncogene product, with [alpha-32P]GTP. Based on our studies, a sensitive, rapid, and specific assay for the detection of multiple forms of ras p21 has been developed. The specificity of this protocol is shown by (a) sensitivity of affinity labeling of ras p21 to known inhibitors of GTP binding and (b) immunoprecipitation of affinity labeled protein with anti-ras p21 serum. Detection and semiquantitation of ras p21 by this method is accomplished in less than 24 h and requires as little as 100,000 cells or about 5 mg of tissue sample from skin tumor, liver, and mammary tumor tissues. Furthermore, using this approach, we were able to detect the selective loss of one species of ras p21 in transplanted Morris hepatoma cells.