Stuart L. Marcus

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.

The relative hydrophobicity of oncornaviral structural proteins

Abstract The interaction of the major structural polypeptides from the murine mammary tumor virus (MuMTV) and Rauscher murine leukemia virus (R-MuLV) with alkyl-agarose derivatives containing hydrocarbon arms of various lengths was examined. Both R-MuLV and MuMTV polypeptides were selectively adsorbed to octylimino (C 8 )-agarose and decylimino (C 10 )-agarose columns. Elution of proteins was accomplished using buffers containing 8.5 M ethylene glycol. In certain cases, polypeptides could only be removed from the columns by the addition of detergent to ethylene glycol-containing buffers. By examining the chromatographic behavior of polypeptides using (C 8 )- and (C 10 )-agarose columns and the conditions required for the elution of adsorbed proteins, we were able to determine the relative potential for hydrophobic interaction (degree of hydrophobicity) for each viral protein. The most hydrophobic polypeptide of MuMTV was found to be the glycoprotein gp34, while p15(E) and the gag precursor prp67 appeared to be the most hydrophobic of the R-MuLV proteins. The least hydrophobic polypeptides of MuMTV were p23 and p16, while those of R-MuLV were the two major internal proteins, p10 and p12. Four degrees of apparent hydrophobicity, from least hydrophobic to strongly hydrophobic, could be discerned by this procedure. The major core proteins (MuMTV p28, R-MuLV p30) of both viruses were slightly hydrophobic, while their major glycoproteins were moderately hydrophobic. The degree of hydrophobicity of oncornaviral polypeptides, as determined by hydrophobic chromatography, appeared to bear some relation to the different subviral components with which the proteins were associated. Our results indicate that hydrophobic chromatography may provide a new parameter for the characterization of oncornaviral polypeptides. This procedure should also prove applicable to the study of polypeptides from other viral systems.

Effects of exogenous polyamine and trypanocides on the DNA polymerase activities from Trypanosoma brucei brucei, mouse thymus and murine leukemia virus.

The effects of exogenously added spermine on activated (gapped) DNA-directed and poly(dC) . (dG)12-18-directed DNA synthesis were tested on the chromatographically separated DNA polymerase activities of Trypanosoma brucei brucei. Activated DNA-directed DNA synthesis by the Peak I (eluting from DNA-agarose at 0.15 M KCl) and Peak II (eluting at 0.3 M KCl) polymerase was consistently inhibited or stimulated, respectively, by exogenous spermine. Kinetic analysis revealed that inhibition of the Peak I enzyme with respect to template DNA occurred by a mixed mechanism, while a major factor in the stimulation of the Peak II enzyme by spermine appeared to be the polyamine-mediated reversal of substrate inhibition by DNA at concentrations above 10 micrograms/ml. The apparent Km values of Peak I and Peak II DNA polymerase for activated DNA were determined to be 5 and 0.5 microgram/ml, respectively. In contrast to the results observed with activated DNA, activation of Peak II-enzyme-catalyzed poly(dC)-directed DNA synthesis was similar at all template-primer concentrations. Peak I enzyme-catalyzed poly(dG) synthesis was either inhibited or slightly stimulated by spermine, depending upon the presence or absence of heteropolymeric DNA, respectively. Dose-dependent inhibition of DNA-directed DNA synthesis catalyzed by T. b. brucei DNA polymerases, murine thymus DNA polymerase alpha, and Rauscher murine leukemia virus reverse transcriptase by trypanocides was examined to determine a possible mechanism of selective toxicity by such agents. The drugs Antrycide (quinapyramine), pentamidine, imidocarb, Berenil (diminazene aceturate), WR-199-385-[2,5-bis(4-guanylphenyl)furan . 2HCl] and isometamidium inhibited DNA polymerases of the eucaryotic cells at approximately the same degree, and at similar concentrations. The presence of spermine in reaction mixtures did not spare any drug inhibition. Stimulation of reverse transcriptase activity was observed in the presence of Antrycide and imidocarb, however, this could be negated by stimulatory amounts of spermine present in the reaction mixture. The results, obtained using an activated DNA-directed assay system, suggest that trypanosomal DNA polymerases are not the selective target of trypanocidal drugs currently available.

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.

Chromatographic resolution of two DNA polymerase activities from bloodstream forms of Trypanosomabrucei: Differential responses to exogenous polyamine addition

Abstract A single peak of DNA polymerase activity from extracts of T. brucei , obtained by DEAE-cellulose and phosphocellulose ion-exchange chromatography, was resolved into two peaks differing in KCl concentration necessary to elute them from a DNA-agarose column. Peak I (eluting at 0.2 M KCl) and Peak II (eluting at 0.4 M KCl), differed in response to increasing KCl concentrations, although both functioned optimally with Mg2+ as divalent cation when DNA synthesis was directed either by activated DNA or poly (dC)·(dG)12–18. Due to the potential significance of polyamines in the metabolism of T. brucei , the effect of exogenous polyamine on rates of DNA synthesis by the peak I and II enzymes was compared with that of murine DNA polymerase alpha. Only the peak I enzyme was significantly stimulated (up to 4-fold) by the biologically active polyamines spermine and spermidine at physiological concentrations. The response of the peak I enzyme resembled that of the alpha polymerase. This result suggests a possible functional difference between peak I and II enzymes, as well as a potential target site for trypanocidal drug development.

Phosphoproteins of the murine mammary tumor virus

Abstract Murine mammary tumor virus (MuMTV) labeled in vivo with [ 32 P]orthophosphate and 3 H-amino acids was analyzed on cylindrical sodium dodecyl sulfate (SDS)-polyacrylamide gels. Of all the MuMTV polypeptides (gp47, gp34, p27, p23, p16, and p12), only two, p23 and p27, appeared to co-migrate with labeled phosphate. The MuMTV structural proteins were fractionated by hydrophobic chromatography (which completely separates p23 from p27) and the resulting fractions were analyzed by both cylindrical and slab gel SDS-polyacrylamide gel electrophoresis to determine the 32 P label associated proteins and for the resistance or sensitivity of the label to RNase and protease. The results of these studies indicated that both p23 and p27 contained phosphate. Fractionation of MuMTV polypeptides performed in an A 5m agarose column in the presence of guanidine-HCl yielded homogenous preparations of p27, which was also found to be associated with phosphate. The major phosphoamino acid of both p23 and p27 was found to be phosphoserine. Immunoprecipitation with monospecific anti-p27 serum showed that p23 is antigenically unrelated to p27. Thus, it appears that MuMTV contains two distinct phosphoproteins. A comparison of the 32 P and 3 H labels of both p23 and p27 showed that more phosphate per amino acid residue is associated with p23 than with p27, suggesting that p23, although a minor polypeptide, is the major phosphorylated component of the virus.

Polyamines stimulate natural RNA-directed DNA synthesis by Rauscher murine leukemia virus DNA polymerase

Abstract In the presence of optimal concentrations of Mg 2+ , spermine and spermidine were found to stimulate rabbit globin mRNA-directed cDNA synthesis by Rauscher murine leukemia virus (R-MuLV) DNA polymerase. Stimulation of DNA synthesis did not occur with the polyamines putrescine or cadaverine, nor could exogenously provided salt or ammonium ions duplicate the stimulation. Analysis of the mechanism of stimulation showed that inclusion of spermine in reaction mixtures a) increased Vmax and decreased apparent Km with respect to the globin mRNA-oligo(dT) temṕlate-primer complex, and b) decreased the quantity of oligo (dT) required for optimal rates of cDNA synthesis on a fixed quantity of mRNA template. Genomic 70S RNA-directed cDNA synthesis was also stimulated by spermine addition to reaction mixtures, but only at supra-optimal RNA concentrations. Our results suggest that stimulation of R-MuLV DNA polymerase activity by polyamines is primarily due to stabilization of the enzyme-templateprimer initiation complex resulting in increased efficiency of initiation of cDNA synthesis.

The mechanism of inorganic phosphate-mediated inhibition of calf thymus DNA polymerase β and Rauscher leukemia virus DNA polymerase

Abstract Calf thymus DNA polymerase β and mammalian type C retroviral DNA polymerases are strongly inhibited by low concentrations (1–2mM) of inorganic phosphate (Pi). A detailed analysis of this phenomenon revealed that Pi-mediated inhibition: a) requires the presence of Mn2+ (Mg2+ neither supports nor relieves this inhibition; b) is strongly affected by the stoichiometric relationship between Mn2+ and Pi concentrations; c) is competitive with respect to deoxynucleoside triphosphate (dNTP) concentration, and d) increasing the concentration of substrate or non-substrate dNTPs in reaction mixtures raised the concentration of Mn2+ at which significant inhibition by a fixed concentration of Pi was first seen. These findings suggested that Mn2+, dNTPs, and Pi may interact in Pi-mediated inhibition. Thin-layer chromatographic analysis revealed the formation of an Mn-dNTP-Pi complex, while Mg2+ did not participate in such complex formation. We propose that it is this tripartite complex which is responsible for the Pi-mediated inhibition of sensitive DNA polymerases.

Hydrophobic interaction of retroviral DNA polymerases with alkyl-agarose matrices

Abstract The chromatographic behavior of DNA polymerase activity in solubilized preparations of Rauscher murine leukemia virus (R-MuLV), murine mammary tumor virus (MuMTV), Mason-Pfizer monkey virus (MP-MV), and avian myeloblastosis virus (AMV) was examined on agarose derivatives containing hydrocarbon arms of various lengths. Ethyl-(C 2 )- and hexylimino-(C 6 )-agarose failed to bind significant quantitites of DNA polymerase activity. R-MuLV DNA polymerase bound to octylimino-(C 8 )- and decylimino-(C 10 )-agarose, and could be quantitatively recovered from these matrices by elution with buffers containing ethylene glycol alone and in the presence of detergents, respectively. The other viral polymerases demonstrated equal or greater affinities for these matrices. The potential for hydrophobic interaction by viral DNA polymerases revealed by our studies provides a mechanism whereby the specific complexing of reverse transcriptase with retroviral structural proteins in the absence of RNA may be explained.