Mukund J. Modak

Synthesis of compositionally unique DNA by terminal deoxynucleotidyl transferase

Studies on the composition and characterization of DNA product(s) synthesized by calf thymus terminal deoxynucleotidyl transferase were performed using homopolymeric single-stranded, calf thymus double-stranded, and native DNA resident in calf thymus chromatin preparations as priming DNA species. Synthesis was carried out using equimolar concentrations of all four deoxynucleoside triphosphates as substrates and Mg2+ or Mn2+ as an effective divalent cation. Irrespective of the nature of the priming DNA or the divalent cation, the DNA product contained 60-70% dGMP residues, 10-15% each of the two pyrimidine residues, and 5-10% dAMP residues. The product synthesized using chromatin DNA as initiator was predominantly single-stranded and its synthesis was resistant to actinomycin D. The predilection of terminal deoxynucleotidyl transferase to synthesize dGMP-rich products on natural or homopolymeric DNA primers suggests that such products may represent biologically important recognition signal sequences.

Purification and properties of murine mammary tumor virus DNA polymerase

Abstract Murine mammary tumor virus (MuMTV) DNA polymerase was purified by affinity chromatography on polycytidylate-agarose followed by phosphocellulose ion-exchange chromatography. The resulting enzyme preparation contained two major polypeptides, of 85,000 and 50,000 daltons as determined by SDS-polyacrylamide gel electrophoresis, and appeared, except for low levels of RNase H, free of DNase and RNase activity. The molecular weight of the purified native enzyme as determined by velocity sedimentation, was approx. 108,000. MuMTV DNA polymerase appears to be a zinc metalloenzyme and requires at least one reduced sulfhydryl group for the expression of catalytic activity. Apparent K m values determined for synthetic template-primers and deoxynucleoside triphosphates were 1 μg/ml and 10–12 μ M , respectively. Examination of the optimal biochemical conditions for DNA synthesis on a variety of template-primers revealed that Mg 2+ was the preferred divalent cation for DNA synthesis. Mn 2+ could partially substitute for Mg 2+ , although it inhibited DNA synthesis when added in the presence of Mg 2+ . MuMTV DNA polymerase exhibited a preference for (dC) n ·(dG) 12−18 among all the synthetic template-primers tested. Activated DNA was preferred as a heteropolymeric template for DNA synthesis when compared with viral 70 S RNA with either endogenous primers or with (dT) 10 as a primer. Rates of heat inactivation of the MuMTV DNA polymerase were found to vary depending upon the template-primer used to measure that inactivation.

Terminal deoxyribonucleotidyl transferase activity in acute undifferentiated leukemia.

Abstract High levels of terminal deoxyribonucleotidyl transferase (TdT) were found in and partially purified from leukocytes obtained from a patient with acute undifferentiated leukemia. The majority of cells lacked lymphoid cell surface markers, suggesting that the disease may have originated from a popoulation of primitive lymphoid progenitor cells. Phosphocellulose chromatography resolved two peaks of TdT activity, and further studies revealed that a) the two forms of TdT possessed identical molecular weights, b) oligonucleotide primer and substrate preferences for the two enzyme forms were identical to those of TdT from calf thymus, and c) DNA polymerase γ was detected as a contaminant in TdT fractions.

Selective inhibition of terminal deoxynucleotidyl transferase (TdT) by adenosine ribonucleoside triphosphate (ATP) and its application in the detection of TdT in human leukemia

Abstract TdT is strongly inhibited in the presence of ATP, while other DNA polymerases, i.e., α, β, γ and reverse transcriptase are resistant to ATP inhibition. This selective inhibition of TdT in the presence of ATP has been used as a diagnostic aid to identify TdT in extracts of leukocytes obtained from acute lymphocytic leukemia (ALL) and chronic myelogenous leukemia (CML) patients.

The artifactual nature of fluoride inhibition of reverse transcriptase and associated ribonuclease H

Abstract Rauscher leukemia virus reverse transcriptase associated polymerization and ribonuclease H activities were totally resistant to sodium fluoride inhibition in contrast to AMV reverse transcriptase enzyme activities. We have found that the observed fluoride mediated inhibition is divalent-cation specific and is solely due to Mg2+ dependent precipitation of substrates and polynucleotides. Furthermore, the differential inhibition of AMV polymerase and RNase H could be explained on the basis of stoichiometry of fluoride to polynucleotide substrate or template primer used in the individual assay. The non-specific nature of fluoride mediated inhibition is further confirmed by the observation that the fluoride sensitivity of several DNA polymerases and RNase H activities is seen only when Mg2+ is an effective divalent cation.

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.

Reverse transcriptase-associated RNase H. Part IV. Pyrophosphate does not inhibit RNase H activity of AMV DNA polymerase

Abstract Sodium pyrophosphate, a known inhibitor of DNA polymerases, strongly inhibits DNA synthesis directed by various synthetic and natural template-primers and catalysed by reverse transcriptases from AMV and RLV, but has no effect on the reverse transcriptase-associated ribonuclease H activity. The synthesis of both actinomycin D sensitive and insensitive DNA under the direction of 70 S AMV RNA or globin mRNA with high or low concentrations of substrate triphosphates is inhibited by the addition of pyrophosphate but under none of these conditions, could RNase H activity be suppressed by inclusion of pyrophosphate. These studies further establish that the site for RNase H function is distinct from the substrate binding site and that the target for pyrophosphate action is not the RNase H (template binding site).

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.

Terminal deoxynucleotidyl transferase (TdT) enzyme in thymus and bone marrow: I. Age-associated decline of TdT in humans and mice

Abstract This study was aimed at characterizing terminal deoxynucleotidyl transferase (TdT) levels in populations of normal human and murine lymphocytes and toward correlating TdT enzyme levels with the biological process of aging. A newly developed method that utilizes a small number of cells was employed to determine TdT levels in bone marrow and thymus cells following cell fractionation at unit gravity sedimentation. By these methods, cell fractions with high TdT activity were found to comprise only 5–10% of the parent cell pools. In the human bone marrow, we show here that TdT-positive cell fractions are largely depleted of HTLA, E-rosette forming, and mitogen-responsive cells, whereas TdT-positive human thymocyte fractions contain a high percentage of HTLA and E-rosette-positive cells. Our observations in the murine model confirm the earlier observations that TdT activity decreases with age. We further show here that the age-associated decline of TdT in the bone marrow preceded that in the thymus. As is true for the mouse, TdT activity in human bone marrow and thymus was also found to decrease with advancing age. The decline in TdT was not associated with a change in cell distribution profiles after unit gravity sedimentation of bone marrow or thymus cells. From these data, the age-associated loss of TdT cannot be attributed to a loss of a particular subpopulation of cells.

Adenosine 5′-triphosphate (ATP)-mediated stimulation and suppression of DNA synthesis in lymphoid cells. II. Suppressive effect of ATP on murine T-cell functions

The suppressive effects of ATP on murine T-cell functions were studied. The suppressive effects of ATP as well as adenosine on the DNA synthesis of spleen cells are due to the presence of mature T-cells, because ATP has no suppressive effect on athymic nu/nu spleen cells. Further characterization of the cells which are responsible for ATP-mediated suppression of DNA synthesis revealed that the cells are nylon wool-adherent T-cells and PHA-reactive T-cells. In addition, the suppressive effects of ATP on both spontaneous and mitogen-induced proliferative responses are stronger than that of adenosine, and T-cells are more sensitive to ATP than B-cells. The observation that both ATP and adenosine have unique effects on T-cells compared to B-cells may contribute toward explaining why patients with severe combined immunodeficiency (SCID) associated with adenosine deaminase (ADA) deficiency have greater T-cell than B-cell abnormalities.