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Detection and characterization of DNA polymerase activity in Toxoplasma gondii

Published online by Cambridge University Press:  06 April 2009

A. Makioka ,
B. Stavros ,
J. T. Ellis  and
A. M. Johnson
A. Makioka
Affiliation:
Department of Microbiology, School of Biological and Biomedical Sciences, University of Technology, Sydney, Gore Hill, New South Wales 2065, Australia
B. Stavros
Affiliation:
Department of Microbiology, School of Biological and Biomedical Sciences, University of Technology, Sydney, Gore Hill, New South Wales 2065, Australia
J. T. Ellis
Affiliation:
Department of Microbiology, School of Biological and Biomedical Sciences, University of Technology, Sydney, Gore Hill, New South Wales 2065, Australia
A. M. Johnson
Affiliation:
Department of Microbiology, School of Biological and Biomedical Sciences, University of Technology, Sydney, Gore Hill, New South Wales 2065, Australia
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Summary

A DNA polymerase activity has been detected and characterized in crude extracts from tachzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+ markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.

Keywords

DNA polymeraseToxoplasma gondiiparasitic protozoa
Type
Research Article
Information
Parasitology , Volume 107 , Issue 2 , August 1993 , pp. 135 - 139
Copyright
Copyright © Cambridge University Press 1993

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References

REFERENCES

Abu-Elheiga, L., Spira, D. T. & Bachrach, U. (1990). Plasmodium falciparum: properties of an α-like DNA polymerase, the key enzyme in DNA synthesis. Experimental Parasitology 71, 21–6.Google Scholar
Chang, L. M. S. & Bollum, F. J. (1972). Antigenic relationships in mammalian DNA polymerase. Science 175, 1116–17.Google Scholar
Chang, L. M. S., Cheriathundam, E., Mahoney, E. M. & Cerami, A. (1980). DNA polymerases in parasitic protozoans differ from host enzymes. Science 208, 510–11.Google Scholar
Choi, I. & Mikkelsen, R. B. (1991). Cell cycle-dependent biosynthesis of Plasmodium falciparum DNA polymerase-α. Experimental Parasitology 73, 93–100.CrossRefGoogle ScholarPubMed
De Vries, E., Stam, J. G., Franssen, F. F. J., Van Der Viet, P. C. & Overdulve, J. P. (1991). Purification and characterization of DNA polymerase from Plasmodium berghei. Molecular and Biochemical Parasitology 45, 223–32.CrossRefGoogle ScholarPubMed
Dube, D. K., Williams, R. O., Seal, G. & Williams, S. C. (1979). Detection and characterization of DNA polymerase from Trypanosoma brucei. Biochimica et Biophysica Acta 561, 10–16.Google Scholar
Fry, M. & Loeb, L. A. (1986). Animal Cell DNA Polymerases. Boca Raton Florida: CRC Press.Google Scholar
Holmes, A. M., Cheriathundam, E., Kalinski, A. & Chang, L. M. S. (1984). Isolation and partial characterization of DNA polymerases from Crithidia fasciculata. Molecular and Biochemical Parasitology 10, 195–205.Google Scholar
Johnson, A. M., Makioka, A. & Ellis, J. T. (1993). A strategy for cloning a DNA polymerase gene of Toxoplasma gondii. In Toxoplasmosis, NATO/ARW Series (ed. Smith, J.). Berlin, Heidelberg: Springer-Verlag. (in the Press.)Google ScholarPubMed
Kesti, T. & Syväoja, J. E. (1991). Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase σ. journal of Biological Chemistry 266, 6336–41.Google Scholar
Leegwater, P. A. J., Strating, M., Murphy, N. B., Kooy, R. F., Van Der Vliet, P. C. & Overdulve, J. P. (1991). The Trypanosoma brucei DNA polymerase α core subunit gene is developmentally regulated and linked to a constitutively expressed open reading frame. Nucleic Acids Research 23, 6441–7.Google Scholar
Nolan, L. L. & Rivera, J. H. (1991). Partial purification and characterization of a β-like DNA polymerase from Leishmania mexicana. Biochemistry International 25, 499–508.Google Scholar
Nolan, L. L. & Rivera, J. H. (1992). The DNA polymerases of Leishmania mexicana. FEMS Microbiology Letters 95, 71–6.Google Scholar
Nolan, L. L., Rivera, J. H., Khan, N. N. (1992). Isolation and partial characterization of a high-molecular weight DNA polymerase from Leishmania mexicana. Biochimica et Biophysicia Acta 1120, 322–8.Google Scholar
Pfefferkorn, E. R. (1984). Characterization of a mutant of Toxoplasma gondii resistant to aphidicolin. journal of Protozoology 31, 306–10.CrossRefGoogle ScholarPubMed
Ridley, R. G., White, J. H., McAleese, S. M., Goman, M., Alano, P., De Vries, E. & Kilbey, B. J. (1991). DNA polymerase δ: gene sequences from Plasmodium falciparum indicate that this enzyme is more highly conserved than DNA polymerase α. Nucleic Acids Research 19, 6731–6.Google Scholar
Rojas, C., Venegas, J., Litvak, S. & Solari, A. (1992). Two DNA polymerases from Trypanosoma cruzi: biochemical characterization and effects of inhibitors. Comparative Biochemistry and Physiology 101C, 27–33.Google Scholar
So, A. G. & Donkey, K. M. (1988). Mammalian DNA polymerases α and δ: current status in DNA replication. Biochemistry 27, 4591–5.CrossRefGoogle ScholarPubMed
Solari, A., Tharaud, D., Repetto, Y., Aldunate, J., Morello, A. & Litvak, S. (1983). In vitro and in vivo studies of Trypanosoma cruzi DNA polymerase. Biochemistry International 7, 147–57.Google Scholar
Tanaka, S., Hu, S.-Z., Wang, T. S-F. & Korn, D. (1982). Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase α. Journal of Biological Chemistry 257, 8386–90.Google Scholar
Torri, A. F. & Englund, P. T. (1992). Purification of a mitochondrial DNA polymerase from Crithidia fasciculata. Journal of Biological Chemistry 267, 4786–92.Google Scholar
Tsurimoto, T., Melendy, T. & Stillman, B. (1990). Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature, London 346, 534–9.Google Scholar
Wang, T. S-F. (1991). Eukaryotic DNA polymerases. Annual Review of Biochemistry 60, 513–52.Google Scholar