Isolation and partial characterization of a high-molecular-weight DNA polymerase from Leishmania mexicana.
This paper describes for the first time the isolation and characterization of a high-molecular-weight predominant DNA polymerase from the genus Leishmania, which are parasitic flagellated protozoa. Like mammalian DNA polymerase alpha, the leishmanial DNA polymerase, designated DNA polymerase A, is of high-molecular-weight, is sensitive to N-ethylmaleimide and is inhibited by high ionic strength. Unlike mammalian DNA polymerase alpha, but similar to the predominant DNA polymerase isolated from the related lower eukaryotic organisms, Trypanosoma cruzi and Crithidia fasciculata, the leishmanial DNA polymerase A is resistant to inhibition by aphidicolin, a potent inhibitor of DNA replication in mammalian cells and of DNA polymerase alpha. The DNA polymerase A was purified 28,000-fold and properties such as pH optimum, salt sensitivity, template requirements and response to DNA polymerase inhibitors were determined. A low-molecular-weight DNA polymerase was detected during the isolation procedures, but was separated from the polymerase A activity. Differences in responses to specific antisera and specific mammalian DNA polymerase alpha inhibitors suggest that the leishmanial high-molecular-weight A enzyme is sufficiently different to suggest this enzyme as a chemotherapeutic target.
Nolan
L L
LL
School of Public Health, University Massachusetts, Amherst 01003.
Rivera
J H
JH
Khan
N N
NN
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000302:isolation & purification*; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D007525:Isoelectric Focusing; D007894:Leishmania mexicana / Q000201:enzymology* / Q000235:genetics; D008970:Molecular Weight; D019384:Nucleic Acid Synthesis Inhibitors; D009994:Osmolar Concentration; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D013698:Templates, Genetic
D019384:Nucleic Acid Synthesis Inhibitors; D015800:Protozoan Proteins; D016590:Aphidicolin; D004259:DNA-Directed DNA Polymerase