Biochemical and molecular biological studies on DNA polymerase of Toxoplasma gondii

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A DNA polymerase activity has been detected and characterized in crude extracts from tachyzoites of virulent RH strain of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6.4 S,corresponding to an approximate molecular weight of 150,000. Like mammalian alpha-type DNA polymerases, the T.gondii enzyme 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 polymerase alpha, delta and epsilon, ant the activity was inhibited by ddTTP which is an inhibitor of mammalian DNA polymerase beta and gamma. Mg^<2+> was required for activity and a higher concentration of K^+ markedly inhibited the activity. Monoclonal antibodies against human DNA polymerase alpha did not bind to the T.gondii enzyme. Thus the T.gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs. The activity of virulent RH strain was siginificantly higher than that of avirulent ME49 strain, suggesting that this increased activity of the enzyme of virulent strain contributes to a faster rate of multiplication of the organisms as aompared with that of avirulent strain. For cloning of DNA polymerase alpha gene of T.gondii, an oligonucleotide probe which included the conserved bases was synthesized and used for screening a T.gondii genomic DNA library in lambda GEM.One positive clone was obtained. The insert DNA was analyzed, found to be 15 kb in size, and then subcloned into pUC18 for sequencing. For cloning of DNA polymerase delta gene of T.gondii, a cDNA fragment was produced by RT-PCR and then sequenced and confirmed as the real gene. A T.gondii cDNA library in lambda gt 10 was screened using this cDNA fragment and two slones were identified and their insert DNAs were subcloned into pBluescript for sequencing.